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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3662-3668
PLENARY PAPER
From the Abteilung Transfusionsmedizin, Universitätsklinikum
Ulm, and DRK-Blutspendedienst Baden-Württemberg, Institut Ulm,
Ulm, Germany.
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
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 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 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 Blood samples and DNA isolation
Yeast artificial chromosome DNA
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 ).
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.
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).
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).
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.
Localization of the RHD gene deletion in 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).
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.
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
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.
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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Top
Abstract
Introduction
, which can be specifically detected by a 37-bp insertion
in exon 4;
trait in a white patient with the RhCCee phenotype attributed to a four-nucleotide deletion in the RHD gene.
Blood.
1998;92:1839
