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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3662-3668
PLENARY PAPER
RHD gene deletion occurred in the Rhesus
box
Franz F. Wagner and
Willy A. Flegel
From the Abteilung Transfusionsmedizin, Universitätsklinikum
Ulm, and DRK-Blutspendedienst Baden-Württemberg, Institut Ulm,
Ulm, Germany.
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Abstract |
The Rh blood group antigens derive from 2 genes,
RHD and RHCE, that are located at
chromosomal position 1p34.1-1p36 (chromosome 1, short arm, region 3, band 4, subband 1, through band 6). In whites, a cde haplotype with a
deletion of the whole RHD gene occurs with a frequency of
approximately 40%. The relative position of the 2 RH genes and
the location of the RHD deletion was previously unknown. A
model has been developed for the RH locus using RHD- and RHCE-related nucleotide sequences deposited in nucleotide sequence databases along with polymerase chain reaction (PCR) and
nucleotide sequencing. The open reading frames of both
RH genes had opposite orientations. The 3' ends of the
genes faced each other and were separated by about 30 000 base pair
(bp) that contained the SMP1 gene. The RHD gene was
flanked by 2 DNA segments, dubbed Rhesus boxes, with a length
of approximately 9000 bp, 98.6% homology, and identical orientation.
The Rhesus box contained the RHD deletion occurring
within a stretch of 1463 bp of identity. PCR with sequence-specific
priming (PCR-SSP) and PCR with restriction fragment length polymorphism
(PCR-RFLP) were used for specific detection of the RHD
deletion. The molecular structure of the RH gene locus explains
the mechanisms for generating RHD/RHCE hybrid alleles and
the RHD deletion. Specific detection of the RHD genotype is now possible.
(Blood. 2000;95:3662-3668)
© 2000 by The American Society of Hematology.
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Introduction |
The Rhesus D antigen (ISBT 004.001; RH1) is the most
important blood group antigen determined by a protein. Anti-D
remains the leading cause of hemolytic disease
of the newborn.1,2 Depending on the population,
3%-25% of whites lack the antigen D.3 Anti-D
immunizations can occur readily in D-negative recipients.4
The antigens of the Rh blood group are carried by proteins coded by 2 genes, RHD and RHCE, that are located at chromosomal position 1p34.1-1p36 (chromosome 1, short arm, region 3, band 4, subband 1, through band 6),5,6 probably within less than a
450 000-base pair (bp) distance.7 Both genes encompass 10 exons, and their structures are highly homologous. Until recently, the
relative orientation of the genes, their distance, and the possibility
of other interspersed genes were unknown.8 Very recently,
Okuda et al9 reported a sequence of about 11 000 bp, which
was thought to represent the DNA segment between RHD and
RHCE.
In whites, the vast majority of D-negative haplotypes is due to a
deletion of the RHD gene. This deletion spans the whole RHD
gene because RHD-specific sequences ranging from exon 1 to the 3' untranslated region are absent.10 The exact
extent of the deletion was uncertain, leaving open the possibility that neighboring genes were also affected.
Identification of the RHD gene as the molecular basis of the D
antigen enabled the RhD phenotype prediction by DNA
typing.8,11 However, because the structure of the prevalent
D-negative haplotype is unknown, a specific detection of the
RHD deletion remained impossible, and discrimination of
RHD+/RHD+ homozygous
individuals from
RHD+/RHD heterozygous
individuals relied on indirect methods. This discrimination is of
particular clinical interest in D-negative mothers with an anti-D: the
risk of an affected child is 100% with an
RHD+/RHD+ father, but it is
only 50% with an
RHD+/RHD father.
Several indirect approaches have been applied to determine zygosity:
(1) A simple guess based on the phenotype is correct in about 95% of
all cases; (2) determination of the D antigen density, which can be
confounded by factors such as the presence of the C antigen; and (3)
several methods involving the parallel quantitative amplification of
RHD- and RHCE-specific sequences.12,13 These elaborate techniques may not be practical in routine
laboratories, however. In addition, several investigators identified
polymorphisms in the RHCE gene or neighboring sequences that
were genetically linked to lack of the RHD
gene.7,14-16 This indirect approach relied on the linkage
disequilibrium associating RHD with a polymorphism.
The most direct approach would be polymerase chain reaction (PCR)
amplification spanning the RHD deletion site. Such an
assay was not available because the structure of the
RHD locus in D-positive and D-negative individuals
was incompletely understood. We developed and proved a model of the
RH gene locus, identified the RHD deletion site in the prevalent D-negative haplotypes in whites, and devised PCR methods for the discrimination of
RHD+/RHD+ and
RHD+/RHD individuals.
Thus, direct testing for the presence of the RHD deletion is now routinely feasible.
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Materials and methods |
Blood samples and DNA isolation
Blood samples anticoagulated with ethylenediamine tetraacetic acid
(EDTA) or citrate were collected from white blood donors. DNA was
isolated by a modified salting-out procedure as described previously.10
Yeast artificial chromosome DNA
DNA from the yeast artificial chromosome (YAC) 38A-A10 (UK HGMP
Resource Center, Cambridge, England) was isolated after a single growth
phase by standard methods.17 We confirmed that this YAC
contained RH DNA. Furthermore, shotgun cloning experiments indicated that some of its insert probably derived from the X chromosome (data not shown).
DNA database searches
The GenBank18 and the chromosome 1 database of the
Sanger Center19 were searched with a complementary DNA
(cDNA) sequence representative of RHD (RhXIII,
accession number X63097) and RHCE (RhVI, accession number
X63095) using the Basic Logical Alignment Search Tool (BLAST) program.
Identification of an RHD-specific sequence in the
RHD promoter
An approximately 2000-bp RHD promoter sequence was
established by chromosomal walking (GenomeWalker kit; Clontech,
Heidelberg, Germany). D-positive and D-negative samples were amplified
using primers re04 and re11d (Table 1), and
RHD- and RHCE-specific sequences were established for
1200 bp 5' of the start codon by sequencing with internal
primers. A short deletion in the RHD gene was identified and
used to develop the RHD-specific primer re011d. The
1200-bp sequence, including the RHD promoter, was deposited at
EMBL under accession number AJ252314.
PCR
If not mentioned otherwise, PCR reactions comprised 60°C
annealing, a 10-minute extension at 68°C, and denaturation at
92°C using the expand long-template or the expand high-fidelity PCR systems (Boehringer Mannheim, Mannheim, Germany) and the listed primers
(Table 1). We used 3 PCR reactions to bridge gaps in the 3'
flanking regions of the RH genes. PCR 1 was completed using primers rea7 and rend31; PCR 2, rend32 and sf1c; and PCR 3, rea7 and
sf3. The structure of the 5' flanking regions was confirmed with
PCR amplifications involving sense primers rend32, rey14a, and rey15a
and antisense primers re011d and re014. The intron 9 size was estimated
to be about 9000 bp, based on PCR amplifications using rb10b and rr4
for RHD (re96 and rh7 for RHCE).
Nuleotide sequencing
Nucleotide sequencing was performed with a DNA sequencing unit
(Prism BigDye terminator cycle-sequencing ready reaction kit and ABI
373A; Applied Biosystems, Weiterstadt, Germany).
Evaluation of the genomic structure of SMP1
The sizes of the SMP1 introns were estimated by PCR
amplicons obtained with primers rend32, sr9, sf1c, sf1, sm19, sr45,
sr47, sr47c, sr5, sr5c, sr55, sr55c, sr3, sr3kp, and rea7.
The positions of the intron/exon junctions and the
absence of additional introns were determined by nucleotide sequencing.
Long-range PCR-SSP to specifically detect the RHD
deletion
PCR was performed using the expand long-template PCR system with
buffer 3 and primers rez4 (5' of upstream Rhesus box) and sr9 (SMP1 exon 1). Annealing was at 60°C, with a 20-minute
extension at 68°C. PCR amplicons were resolved using a 1% agarose gel.
PCR-RFLP to detect the RHD deletion
PCR was performed using the expand high fidelity PCR system and
primers rez7 (nonspecific, 5' of Rhesus box
identity region) and rnb31 (specific for downstream Rhesus box,
3' of downstream Rhesus box identity region). Annealing
was at 65°C, and extension was for 10 minutes at 68°C. PCR
amplicons were digested with PstI for 3 hours at 37°C, and
fragments were resolved using a 1% agarose gel.
Sequencing of the Rhesus boxes
The Rhesus boxes were amplified and sequenced using internal
primers in 2 overlapping fragments with PCR primer pairs
rez4/rend31 and rend32/re011d (upstream Rhesus box),
rea7/rend31 and rend32/sr9 (downstream Rhesus box),
and rez4/rend31 and rend32/sr9 (hybrid Rhesus box of
RHD).
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Results |
DNA database searches and analysis
The high throughput sequences of the GenBank and the chromosome 1 database of the Sanger Center were screened for nucleotide sequences
homologous to RHD or RHCE cDNA. We identified the
84 810-bp genomic clone dJ469D22 (GenBank accession number AL031284), the 129 747-bp genomic clone dJ465N24 (GenBank accession number AL031432), and the 2234-bp SMP1 cDNA (GenBank accession number AF081282). The genomic clone dJ469D22 represented a major fragment of
the RHCE gene, starting 33 340-bp 5' of the RHCE
start codon and ending 1142-bp 3' of exon 9. In dJ465N24, an
internal stretch of 1418 bp located between positions 120 158 and
121 568 was 96% homologous to the 3' end of the RHD
cDNA. The 3' end of the SMP1 cDNA was complementary to
the 3' end of the RHCE cDNA, with an overlap of 58 bp.
The RH gene locus
We derived a physical structure of the RH gene locus (Figure
1) by reviewing 3' and 5'
flanking regions and analyzing YAC 38A-A10, as described in the
paragraphs that follow.
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| Fig 1.
Schematic structure of the RH gene locus.
(A) The positions and orientations of the genes and the Rhesus
boxes are indicated by open arrows and triangles, respectively.
The exons are shown as vertical bars, and their exon numbers are
indicated. The 2 RH genes have opposite orientation, face each
other with their 3' ends, and are separated by about 30 000 bp.
A third gene, SMP1, has the same orientation as RHD and
is positioned between RHD and RHCE. The RHD
gene is flanked on both sides by the 2 highly homologous Rhesus
boxes, which are noted by (b). All exons are
shorter than 200 bp, with the exception of the RHD and SMP1
3' terminal exons. (B) Data used to establish this structure
include the extension of genomic sequences represented in cDNA
(horizontal arrows) and identities and homologies to genomic clones as
noted: bar a, identity with dJ465N24; bar b, homology of RHD to
dJ469D22; bar c, homology of RHD 3' part to dJ465N24; and
bar d, identity with dJ469D22. The positions of 3 bridging PCR
reactions are indicated. The correct position of a nucleotide stretch
previously reported by Okuda et al9 as a "spacer"
sequence between RHD and RHCE is indicated by the bar
labeled with the letter "s."
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3' flanking region.
The 3' flanking region of RHD was highly homologous to
the 3' part of the genomic clone dJ465N24 (Figure 1B, region c).
This homology continued beyond the end of the RHD cDNA and
extended for at least 8000 bp, as proven by the fact that it was
possible to obtain PCR amplicons (Figure 1B, PCR 1). Sequences
homologous to the 3' part of genomic clone dJ465N24 were
neighboring to the 5' region of the SMP1 gene (Figure 1B,
PCR 2). The 3' end of the SMP1 gene occurred immediately
adjacent to the RHCE gene, as indicated by the complementarity
of the 3' ends of the respective cDNAs and confirmed by PCR
(Figure 1B, PCR 3). Further details of the RHD 3'
flanking region (Rhesus box) and the SMP1 gene are
described in subsequent paragraphs.
5' flanking region.
The genomic clone dJ469D22 comprised the 33 340-bp 5' flanking
region of RHCE. For RHD, a 466-bp homology between the
3' end of clones dJ465N24 and dJ469D22 indicated that clone
dJ465N24 might represent the 5' flanking sequence of RHD.
We proved this assumption by PCR (Figure
2).
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| Fig 2.
Chromosomal organization of the DNA regions located
5' to the RHD and RHCE genes.
(A) The proposed structure of the RHCE and RHD 5'
flanking regions is depicted. A total of 4941 bp immediately 5'
of the ATG start codons are homologous between the RHCE and
RHD genes (vertically hatched bars). No homology is present
beyond this homology region (diagonally hatched bars). We used 2 genomic clones, dJ469D22 and dJ465N24, for primer design. DJ469D22
comprises the full length of the depicted RHCE region, whereas
dJ465N24 extends only 466 bp into the homology region. The positions of
several PCR primers are indicated: a, rey14a; b, rend32; c, rey15a; d,
re014; and e, re011d. (B) This proposed structure is supported by
several PCR reactions. Forward priming was done with primer a
(RHCE specific, lane 1-3), primer b (RHD specific, lane
4-6), and primer c (RHCE and RHD homology region, lane
7-9). Amplicons were lacking for primer a with RHD-specific
reverse primer e (lane 2) and for primer b with
RHD DNA (lane 6). The other 7 PCR reactions
yielded amplicons of the predicted sizes in accordance with the genomic
structure shown in panel A.
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YAC 38A-A10.
This YAC had been known to contain RHCE exons 2-10 and
RHD exons 1-107 and was thus expected to contain
the DNA segments interspersed between RHD and RHCE. We
checked for the presence of DNA segments representative of different
parts of the RH locus (Table 2), and the results were concordant with the proposed structure of the
RH locus shown in Figure 1A.
SMP1 gene
The genomic structure of the SMP1 gene was evaluated by PCR
using internal primers and nucleotide sequencing (Figure
3). We identified 6 introns. Exon 1 contained 5' untranslated sequences only and was separated from
the Rhesus box by 15 bp. The long 3' untranslated
sequence of exon 7 overlapped with RHCE exon 10. The total gene
size was estimated to be 20 000 bp, therefore resulting, in
conjunction with the downstream Rhesus box, in approximately a
30 000-bp distance between RHD and RHCE (Figure 1).
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| Fig 3.
Chromosomal organization of the SMP1 gene.
The SMP1 gene has 7 exons, and the positions and approximate
sizes of the introns are shown. The start of the published cDNA
(GenBank accession number AF081282) is separated by 15 nt from the
downstream Rhesus box. Exon 1 contains only a 5'
untranslated sequence, and the SMP1 start codon is located in
exon 2. Exon 7 contains 16 codons and 1656-bp 3' untranslated
sequences and is contiguous with the 3' untranslated sequence of
RHCE exon 10.
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Rhesus boxes
Two DNA segments of approximately 9000 bp, located 5' and
3' of the RHD gene, were designated "Rhesus
boxes." They were highly homologous and had identical
orientation (Figure 4). The upstream Rhesus box (5' of RHD) was approximately 9142-bp
long and ended approximately 4900-bp 5' of the RHD start
codon. The downstream Rhesus box (3' of RHD) was
9145-bp long and originated 104 bp after the RHD stop codon.
The Rhesus boxes exactly embraced the part of
RHD with homology to RHCE. The central
portion of both Rhesus boxes contained a nearly
complete remnant of a transposon-like human element (THE-1B). However,
the single open reading frame usually found in the THE-1B element was
abolished due to several nucleotide aberrations occurring in both
Rhesus boxes in parallel, including a nonsense mutation
in codon 4. While there was an overall 98.6% homology between both
Rhesus boxes, a 1463-bp "identity region" located
between positions 5701 and 7163 bp was completely identical, with the
single exception of a 4-bp T insertion in a poly T tract.
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| Fig 4.
Chromosomal organization of the Rhesus boxes.
The physical extension of the upstream Rhesus box (5' to
RHD) is 9145 bp (darkened bar). About 63% of the boxes'
nucleotide sequence consists of repetitive DNA; the types of the repeat
families are indicated. The overall homology between the upstream and
downstream Rhesus box is 98.6%, but within a 1463-bp identity
region (horizontal arrows), there is only a single 4-bp insertion
(double vertical line). A CpG island (double-headed arrow) is located
at the 3' end and is in the downstream Rhesus box
(3' to RHD) adjacent to the SMP1 promoter.
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Localization of the RHD gene deletion in RHD
haplotypes
We reasoned that the homology of the 2 Rhesus boxes
may have been instrumental in the RHD deletion mechanism in the
common RHD haplotypes. We determined the
nucleotide sequence of the Rhesus box in
RHD DNA (Figure
5). The single Rhesus box detected
in the RHD haplotypes had a hybrid
structure. The 5' end of this Rhesus box represented an
upstream Rhesus box, and the 3' end represented a
downstream Rhesus box. We determined that the 903-bp breakpoint region of the RHD deletion was located in the identity region of the Rhesus boxes (Figure 4, arrow pointing to
left).
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| Fig 5.
RHD gene deletion in the Rh-negative haplotypes.
Three of the 3100-bp segments of the Rhesus boxes are
shown. The upper line indicates the nucleotide sequence of the upstream
Rhesus box in D-positive haplotypes; the lower
line indicates the nucleotide sequence of the downstream Rhesus
box in D-positive haplotypes. The middle line gives the nucleotide
sequence of the single Rhesus box carried by Rh-negative
haplotypes. The asterisk symbols denote identical nucleotides. The
RHD deletion occurred in a 903-bp segment of absolute identity
that was part of a 1463-bp identity region. The positions of primers
rez7 and rnb31 are shown (m indicates mismatch).
PstI restriction sites are indicated by upward
carets. The 3 Rhesus boxes were deposited at EMBL under
accession numbers AJ252311 (upstream Rhesus box), AJ252312
(downstream Rhesus box), and AJ252313 (hybrid Rhesus
box).
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Specific detection of the RHD deletion by PCR
We developed 2 PCR-based methods for specific detection of the
RHD gene deletion occurring in the prevalent
RHD haplotypes (Figure
6). These techniques allowed the ready and direct detection of the common RHD
haplotypes, even if they were in trans to
RHD+ haplotypes. We applied PCR-RFLP to a larger
number of samples (Table 3). As expected,
all 33 samples with known genotype were correctly typed. In 68 additional samples representative of the most common phenotypes, our
results were consistent with the known haplotype frequencies in the
population.
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| Fig 6.
Two technical procedures for specific detection of the
RHD deletion in the common RHD
haplotypes.
(A) A long-range PCR amplification with primers located in
non-Rhesus box sequences and (B) PCR-RFLP with primers located
in the Rhesus boxes are shown. The deduced genotypes
are indicated. The primers of the long-range PCR were located 5'
of the upstream Rhesus box (primer rez4) and in SMP1
exon 1 (primer sr9). RHD haplotypes were
detected specifically (panel A, lanes 1-6). DNA homozygous for the
RHD gene was negative because PCR cannot amplify the 70 000-bp
DNA stretch of the RHD gene. For the PCR-RFLP method, the PCR
amplicons (primer rez7 and rnb31) were digested with
PstI. In D-negative haplotypes, there are 3 PstI sites in the amplicon (Figure 5) resulting
in fragments of 1888 bp, 564 bp, 397 bp, and 179 bp (lanes 1-3). The
downstream Rhesus box of D-positive haplotypes lacks 1 PstI site, resulting in fragments of 1888 bp,
744 bp, and 397 bp (lanes 7-9).
RHD+/RHD heterozygotes
show both fragments of 744 bp and 564 bp (lanes 4-6). The
564-bp fragment appears weaker because heterodimers are not cut
by PstI. Primer rnb31 does not amplify the
upstream Rhesus box of D-positive haplotypes.
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Discussion |
The 2 genes, RHD and RHCE, had opposite orientation
and faced each other with their 3' ends. The RHD gene was
surrounded by 2 highly homologous Rhesus boxes. The
physical distance between RHD and RHCE was 30 000 bp
and was filled with a Rhesus box and the SMP1 gene. The
breakpoints of the RHD deletion in the prevalent RHD haplotypes were located in the 1463-bp
identity region of the Rhesus boxes. We established
technical procedures for specifically detecting the
RHD gene deletion in the common RHD haplotypes.
Based on the structure of the RH gene locus (Figure 1), we
propose a parsimonious model for the RHD gene deletion event
(Figure 7). The RHD deletion may be
explained by unequal crossing-over triggered by the highly homologous
Rhesus boxes embracing the RHD gene. The 903-bp
breakpoint region in the Rhesus boxes was located in a
1463-bp stretch of 99.9% homology resembling a THE-1B and an L2
repetitive DNA element (Figure 4). Interestingly, the DNA segment with
more than 60 000 bp, which was deleted in the RHD haplotype, consisted only of
and contained all sequences that were duplicated in the
RHD+ haplotype.
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| Fig 7.
Model of the proposed mechanism causing the prevalent
RHD haplotypes in whites.
(A) The physical structure of the RHD and RHCE gene
locus. (B) An unequal crossing-over between the upstream and downstream
Rhesus boxes can be triggered by their high homology.
The breakpoint region in the Rhesus boxes was
found to be of 100% homology for 903 bp (Figure 5). (C) Resolving
the crossed-over chromosome yields the RH gene
structure of the extant RHD
haplotype.
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Previously, the discrimination of RHD homozygote individuals
from RHD heterozygote individuals was difficult because the
prevalent RHD allele could not be detected
specifically.8,12 Our results provide the basis for
detecting the prevalent RHD haplotypes, and
hence, true RHD genotyping is now feasible.8 We
describe a PCR-RFLP method and a long-range PCR method using either
Rhesus box sequences or Rhesus box flanking sequences. By using the same DNA stretches or combinations thereof, other methods,
such as PCR-SSO or biochips, can be developed.
RHD genotyping by detection of the prevalent
RHD haplotype may not detect some
RHD+ D-negative alleles. In whites, such
alleles are exceedingly rare,8 but they may occur due
to nonsense mutations (eg, RHD(Q41X)),20 deletions (eg, RHD(488del4)21), or
RHD-CE-D hybrid genes (eg, RHD-CE(2-9)-D22
and RHD-CE(4-7)-D).23 In contrast, in
Africans, there are 3 prevalent D-negative alleles: (1) the
RHD deletion; (2) an RHD pseudogene designated
RHD , which can be specifically detected by a 37-bp insertion
in exon 4;24 and (3) an RHD-CE-D hybrid
gene25,26 associated with the CdeS haplotype.
Based on the data given by Singleton et al,24 these alleles
represent 43%, 43%, and 15% of the D-negative alleles in the black
population, respectively. Hence, all 3 alleles must be specifically
detected for a reliable RHD genotyping in Africans and any other population harboring these alleles. A similar
situation is present in the Japanese. In this population, the
RHD deletion occurs with a frequency of 94% among
D-negative alleles, and additional detection of the
underlying cause of the RHD+ D-negative
alleles associated with RHD(G314V)27 may be warranted.
The opposite orientation of the 2 RH genes explained the
different character of hybrid genes in the MNS and Rh blood group: The
glycophorin genes encoding the MNSs antigens occur in the same orientation,28 and many recombinations may be
explained as an unequal crossing-over resulting in single hybrid
genes.29 In the RH locus, the inversely oriented
sequences are unlikely to trigger unequal crossing-over, and if this
event occurred, no functional hybrid gene would result. Our conclusion
that unequal crossing-over at the RH gene locus was unlikely
may explain that most RH hybrid genes are of either the
RHD-CE-D or RHCE-D-CE type and involve stretches of
homologous DNA positioned in cis, as noted by us
previously.30 Currently, the RH gene system is the only well investigated gene locus where the 2 genes have opposite orientation, rendering it a model system for the evolution
of neighboring, oppositely oriented genes that are frequent
throughout genomes.
Surprisingly, our data show that 3 genes are located at the
RH locus: RHD, RHCE, and SMP1. The
nucleotide sequence of the latter gene has been deposited in the
GenBank as a putative member of an 18-kd small membrane protein family,
and its function is as yet unknown. The gene shows homology to an open
reading frame on chromosome 21.31 Its position between both
RH genes implies that any polymorphism of the
SMP1 gene would be tightly linked to a specific RH
haplotype. It might be anticipated that functionally relevant
mutations of the SMP1 gene may cause selection
pressure for or against specific RH haplotypes. Such
factors might explain some previously unresolved issues of RH
haplotype distribution, such as the high frequency of
RH in the European population. Screening for
polymorphisms in SMP1 appears to be necessary to further
understand the RH locus.
While the molecular mechanism resulting in the prevalent
RHD haplotype is apparent, it is less clear
how the much older duplication event gave rise to the structure of the
RH genes in RHD+ individuals. The
duplication of the Rhesus box and the RH genes probably
occurred as a single event, because the overall homology of the 2 Rhesus boxes is very similar to that of the RH
genes. It is tempting to speculate that the RHD duplication
originated in a causal connection with the insertion of the near
full-length transposon-like THE-1B in duplicate. However, the open
reading frame of the THE-1B probably was nonfunctional at the time of the duplication. Further characterization of related RH gene
loci, including those of the monkey species, will be helpful to resolve the duplication mechanism. Although the structure of the RH
locus in RHD haplotypes is probably very
similar to the structure predating the RH gene duplication, the
"original" RH gene at the current RH chromosomal
position is not extant in the human species. The number of RH
genes is variable among primates,32 and unequal crossing-over may explain both the loss of RHD and the possible generation of more than 2 RH genes in some species.
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Acknowledgments |
We thank Marianne Lotsch, Anita Hacker, Sabine Kaiser, Katharina
Schmid, and Sabine Zahn for expert technical assistance and Bernd
Widder for supplying 4 cDE/cDe samples identified in his thesis work.
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Footnotes |
Submitted November 18, 1999; accepted February 8, 2000.
Supported by Project 531 and Project 442 from the
Universitätsklinikum Ulm, Institut Ulm, Ulm, Germany, and by the
DRK-Blutspendedienst Baden-Württemberg, Stuttgart, Germany.
The nucleic acid sequence data were deposited in the European
Molecular Biology Laboratory, Heidelberg, Germany; GenBank, National
Center for Biotechnology Information, Bethesda, MD; and the DNA Data
Bank of Japan, National Institute of Genetics, Mishima, Japan, under
accession numbers AJ252311, AJ252312, AJ252313, and AJ252314.
Reprints: Willy A. Flegel, Abteilung
Transfusionsmedizin, Universitätsklinikum Ulm, and
DRK-Blutspendedienst Baden-Württemberg, Institut Ulm,
Helmholtzstrasse 10, D-89081 Ulm, Germany; e-mail: waf{at}ucsd.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction
