Legal Medicine 3 (2001) 205212

www.elsevier.com/locate/legalmed

RHC/c genotyping based on polymorphism in

the promoter region of the RHCE gene
Mitsunobu Tanaka a,*, Naoko Yamashita a, Junko Takahashi a, Fumiya Hirayama a,
Eiji Kajii b, Yoshihiko Tani a
a
Department of Research, Osaka Red Cross Blood Center, 2-4-43 Morinomiya, Joto-Ku, Osaka 536-8505, Japan
b
Department of Legal Medicine and Human Genetics, Jichi Medical School, Minamikawachi-machi, Tochigi 329-0498, Japan
Received 21 May 2001; received in revised form 29 June 2001; accepted 2 July 2001

Abstract
Designing of PCR tests for the RHC allele is difficult because of the high DNA sequence homology between RHC and RHD
genes, which differ by only a one-nucleotide substitution at position 48 in exon 1 of the RHCE gene. We sequenced the
promoter region of the RHCE gene, and compared our results with the reported sequence. Genomic DNA was prepared from
blood samples collected from 656 Japanese donors. The DNA segment encompassing the promoter region and exon 1 of the
RHCE gene from 30 donors was amplified by PCR and analyzed by DNA sequencing. Four nucleotide differences between
RHC/c and RHD were found at positions 2468, 2304, 258, and 246. On the basis of the nucleotide differences at positions
2468 (RHCE vs. RHD) and 2292 (RHC vs. RHc), we then developed a novel polymerase chain reaction-restriction fragment
length polymorphism (PCR-RFLP) method for RHC/c genotyping. Analysis of the genomic DNA from the 656 donors revealed
that this method could discriminate RHC from RHc, irrespective of the RHD genotype, with only a few exceptions. The
combination of our system and the intron 2-based PCR-RFLP method previously reported may prove to be more accurate than
either of the methods alone, and therefore, useful and valuable for RHC/c genotyping. q 2001 Elsevier Science Ireland Ltd. All
rights reserved.
Keywords: Rh blood group system; RHCE gene; Promoter region

1. Introduction positive chromosome is composed of two closely

The Rh blood group system comprises numerous
antigens, including the clinically relevant ones D, C,
c, E and e [13], which are carried on homologous but
distinct integral membrane proteins with apparent
molecular weights of 3032 kDa [49]. The Rh poly-
peptides are divided into two different types that share
a high level of homology [2,1013]. Analysis of geno-
mic DNA has revealed that the RH locus of each D-
* Corresponding author. Fax: 181-6-6968-4900.
E-mail address: mitsunobu@mac.com (M. Tanaka).
1344-6223/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 1344-622 3(01)00035-9
linked structural genes. The RHD gene encodes the
D polypeptide and the RHCE gene encodes a poly-
peptide carrying either the C or c antigen together
with the E or e antigen [1418]. In the case of hemo-
lytic disease of the newborn, RHD genotyping is espe-
cially useful to investigate the RhD type of the fetus
before birth [1921]. In addition, as with hemolytic
disease of the newborn, autoimmune hemolytic
anemia and hemolytic transfusion reactions may be
due not only to anti-D, but also to anti-C, -c, -E or -
e. Therefore, it is important to determine the RHC/c
and RHE/e genotypes in these cases [22]. For the RHc,
206 M. Tanaka et al. / Legal Medicine 3 (2001) 205212
RHE, and RHe genotyping, PCR assays have been
reported [2326]. Due to the high DNA sequence
homology between the RHC and RHD genes in
exons 1 and 2, the difficulty of establishing reliable
genotyping methods that can distinguish between
RHD, RHC and RHc has been repeatedly noted
[15,2429].
In this study, we sequenced the promoter region of
the RHCE gene [29], and found three nucleotide
differences between the RHC and RHc genes. On
the basis of these nucleotide differences, we devel-
oped a novel polymerase chain reaction-restriction
fragment length polymorphism (PCR-RFLP) system
for RHC/c genotyping.
2. Materials and methods
2.1. Serological typing of Rh blood group
Serological types of the Rh blood group were deter-
mined by standard hemagglutination tests using Ortho
BioClone human monoclonal antibodies (Ortho Diag-
nostic Systems, Raritan, NJ) as described previously
[30].
2.2. Isolation of genomic DNA
Blood samples were collected from 656 Japanese
subjects. The genomic DNA was isolated from
peripheral blood leucocytes as described by Miller
[31]. In some experiments, DNA was prepared from
whole blood using a DNA isolation kit (QIAGEN,
Hagen, Germany).
2.3. Sequencing of the RHCE gene
The DNA segment encompassing the promoter
region and exon 1 of the RHCE gene was amplified
by PCR. The RHCE-specific primers utilized were
CEPR01 (5 0 -CTGTGTAACTATGAGGAGTCAA-
3 0 ) and TRH2 (5 0 -CTTGATAGGATGCCAC-
GAGCC-3 0 ) (Fig. 1). The thermocycling conditions
were as follows: the first cycle of the PCR was
performed at 958C for 9 min, and then 30 cycles of
1 min at 948C, 2 min at 628C, and 2 min at 728C were
carried out using a DNA thermal cycler (TaKaRa PCR
Thermal Cycler TP3200, TaKaRa Shuzo Co. Ltd.,
Shiga, Japan). The PCR products were then separated
by low-melting-temperature agarose gel electrophor-
esis and subcloned into the pCR2.1-TOPO vector
(TOPO TA Cloning, Invitrogen Corporation, Calsbad,
CA). The cloned fragment was sequenced by the dye-
termination method using THERMO Sequenase
(Amersham Pharmacia Biotech, NJ) and the primers
M13F (5 0 -CTGGCCGTCGTTTTAC-3 0 ) and M13R
(5 0 -CAGGAAACAGCTATGAC-3 0 ). DNA sequen-
cing was performed using an automated system
(Model 373A, Version 1.20, PE Applied Biosystems,
Foster City, CA).
2.4. Amplification and restriction fragment length
polymorphism
Amplification was carried out in a final volume of
20 ml containing 200 mM of each dNTP, 10 mM Tris/
HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1 mg/ml
gelatin, 2 U of AmpliTaq Gold DNA polymerase (PE
Applied Biosystems), and 1 mg of genomic DNA. The
primers were CEPR01 (5 0 -CTGTGTAACTATGAG-
GAGTCAA-3 0 ) and CEPR03r (5 0 -AGAGGG-
CATTCTATTCCTTTGA-3 0 ). The PCR
amplification was performed as described above.
The PCR products were digested with SspI (TaKaRa
Shuzo Co. Ltd.) and analyzed by 3% agarose
(NuSieve GTG agarose, FMC Bio Products, Rock-
land, ME) gel electrophoresis.
2.5. Genotyping of RHC/c and RHE/e using allele-
specific PCR
The RHC-, RHc-, RHE-, and RHe-specific PCR
analyses were performed as previously reported
[30]. The primers used were TRH1 (5 0 -
CGCTGCCTGCCCCTCTGC-3 0 ) and TRH2 (5 0 -
CTTGATAGGATGCCACCAGCC-3 0 ) for RHC,
TRH3 (5 0 -CTTGGGCTTCCTCACCTCAAA-3 0 ) and
TRH4 (5 0 -AAGCCGTCCAGCAGGATTGC-3 0 ) for
RHc, TRH5 (5 0 -TGGCCACGTGTCAACTCTC-3 0 )
and TRH7 (5 0 -CATGCTGATCTTCCTTTGGG- 3 0 )
for RHE, and TRH6 (5 0 -TGGCCACGTGTCA-
ACTCTG-3 0 ) and TRH7 (5 0 -CATGCTGAT-
CTTCCTTTGGG-3 0 ) for RHe. The PCR amplifica-
tion was performed under the following conditions:
30 s at 948C, 30 s at 658C, and 30 s at 728C for 30
cycles using a DNA thermal cycler. The amplified
products were then separated by low-melting-
temperature agarose gel electrophoresis.
 M. Tanaka et al. / Legal Medicine 3 (2001) 205212 207

Fig. 1. Nucleotide sequence of the 5 0 flanking regions RHD gene and RHC (sequence #1)/RHc (sequence #2) gene. The first 120 nucleotides of
exon 1 are shown in uppercase and the initiation codon ATG is underlined. The primers for the sequences, CEPR01 and TRH2, are indicated by
arrows. Three nucleotide substitutions in the promoter region of Rhc G to A at position 2292, C to T at position 2283, and G to A at 2210
are indicated by boxes. The putative cis-regulatory elements GATA-1, SP1, Ets, CACC, and NFE-2 are also indicated by boxes.

2.6. PCR assay for the RHC/c-associated
polymorphism in intron 2
The PCR assay which detects the RHC/c-associated
polymorphism in intron 2 was carried out as described
by Poulter et al. [28] using the following oligonucleo-
tide primers: sense, 5 0 -GTGCCACTTGACTTGG-
GACT-3 0 , and antisense, 5 0 -GTGGAACCCAATG-
CCTCTG-3 0 . The PCR amplification was performed
under the following conditions: 40 s at 958C, 1 min at
608C, and 2 min at 728C for 30 cycles using a DNA
thermal cycler. The PCR products were digested with
PstI at 378C for 2 h and then separated by low-melting-
temperature agarose gel electrophoresis.
3. Results
3.1. Sequence of the promoter region in the RHCE gene
The promoter region of the RHCE gene from an
RhCcEe individual is different from that of RHD at
208 M. Tanaka et al. / Legal Medicine 3 (2001) 205212

positions 2468 (G/A), 2304 (C/T), 258 (G/C), and
246 (A/G) [29]. We first cloned the promoter region
and exon 1 of the RHCE gene from the corresponding
amplified DNA of 30 donors. To amplify the promoter
region of the RHCE gene, but not that of RHD gene,
the 5 0 primer was designed to include the nucleotide at
position 2468 (CEPR01) (Fig. 1). To discriminate
RHC from RHc, a 3 0 primer located at the end of
exon 1 of the RHCE gene was used, since the nucleo-
tide at position 48 in exon 1 is different between RHC
and RHc (C vs. G) [30]. The sequencing of six clones
from each of the 30 donors revealed two types of
sequences. The promoter region of the first type
(sequence #1) was the same as that previously
reported for the RHCE promoter region [28], and its
exon 1 was RHC type. The other type (sequence #2)
showed a high homology to sequence #1, but
possessed three nucleotide substitutions in the promo-
ter region at positions 2292 (G to A), 2283 (C to T),
and 2210 (G to A), as well as one substitution in exon
1 at position 48 (C to G), which corresponds to RHc.
In addition, a complete correlation was observed
between the sequence type and serological phenotype
of the 30 donors, namely, all individuals with CC
phenotype and cc phenotype possessed sequence #1
and sequence #2, respectively, while all those typed as
Cc had both sequences #1 and #2. Taken together,
these findings indicate that sequences #1 and #2 are
derived from the RhC and Rhc genes, respectively.
3.2. Genotyping of RhC/c using PCR-RFLP
Having determined the DNA sequence differences
in the promoter regions of the RHC, RHc, and RHD
genes, we next attempted to develop a promoter
region-based PCR-RFLP system for RHC/c genotyp-
ing. DNA was amplified using the common RHCE
promoter-specific primers (CEPR01 and CEPR03r),
and the PCR products were then digested with SspI.
Since an SspI site is present at position 2292 of the 5 0
flanking region of RHc, but not of RHC, as shown in
Fig. 2A, the 302-bp PCR product of RHc, but not of
RHC, is expected to be digested into 199- and 103-bp
fragments. Using this system, we examined the geno-

Fig. 2. Nucleotide sequence of the RHC/c 5 0 flanking region. The SspI restriction site is indicated by an arrowhead. (A) The primers for RFLP,
CEPR01 and CEPR03r, are indicated by arrows. (B) RhC/c-specific PCR was carried out with primers CEPR01 and CEPR03r and the PCR
products were digested with the restriction enzyme SspI. Lane 1, CCDEe; lane 2, CCDee; lane 3, CcDee; lane 4, CcDEe; lane 5, ccDEE; lane 6,
ccEe; and lane 7, ccee. The PCR assay was carried out in a 20-ml reaction volume, and a 10-ml aliquot was digested with SspI.
 M. Tanaka et al. / Legal Medicine 3 (2001) 205212 209

Table 1
RHC/c alleles with substitutions in exon 1 and the promoter region
of the RHCE gene

Phenotype Genotype Nucleotides and position Number

Exon 1 Promoter
48 2 292

CCDee CC C G 173
CCDEe CC C G 2
CCee CC C G 17
CcDee Cc C/G G/A 22
CcDEe Cc C/G G/A 141 Fig. 3. Allele-specific PCR of RhC/c (A) and SspI cleavage patterns
Ccee Cc C/G G/A 44 of the PCR products of the promoter region (B). Lanes 1, 2 and 5,
CcEe Cc C/G G/A 30 ccEe; lanes 3, 4 and 6, ccee; lanes 1 and 3, RhC-specific PCR; and
ccDEE cc G A 77 lanes 2 and 4, Rhc-specific PCR. Lanes 5 and 6, RhC/c-specific PCR
ccDEe cc G A 2 was carried out with primers CEPR01 and CEPR03r, and the PCR
ccEE cc G A 27 products were digested with the restriction enzyme SspI.
ccEe cc G A 41
ccEe Cc C/G G/A 3
ccee cc G A 63 of all the exceptional individuals yielded the RHC
ccee Cc G/G G/A 14 product (lanes 1 and 3) as well as the RHc product
656 (lanes 2 and 4), indicating that their exon 1 was also
type Cc. The second method we used was a PCR-
mic DNAs of 656 donor samples. RHC/c genotypes RFLP method established by Poulter et al. on the
determined using this system were almost completely basis of the nucleotide polymorphism in intron 2 of
concordant with RhC/c serological phenotypes as the RHD and RHCE genes [28]. The PCR products of
determined by the hemagglutination test (Table 1). Rhc, but not of RHC, are expected to be digested into
The PCR products of all individuals with the cc two fragments (390 and 250 bp) after PstI treatment.
phenotype yielded bands of 199 and 103 bp after The results of two representative cases (ccee and
digestion with SspI, while the amplicon from CC indi- ccEe) are shown in Fig. 4: all 17 cases yielded the
viduals remained uncut after the enzyme treatment. In
addition, those with the Cc phenotype revealed a
combination of the three bands regardless of their
RhD and RhE/e phenotypes (Fig. 2B, lanes 3 and 4).
However, three cases of the ccEe phenotype were
genotyped as Cc and 14 cases of the ccee phenotype
as Cc (Table 1). The representative results of two such
cases (ccee and ccEe) are shown in Fig. 3B. It is
noteworthy to mention that all the exceptional cases
were RhD-negative and serologically C-negative.
None of the RhD-positive ccDee or RhD-positive
CcDEe donors showed such a discrepancy.
The discrepancy between genotype and serological
type observed in the 17 cases prompted us to examine
the genotypes of these cases by other genotyping
methods. We first used a previously established
Fig. 4. PstI cleavage patterns of the PCR products for intron 2. Lane
allele-specific PCR method based on sequence differ-
1, ccee; lane 2, ccEe; and lane 3, ccDee. RHC/c-specific PCR was
ences in exon 1 at position 48 and in exon 2 at posi- carried out with primers: sense, 5 0 -GTGCCACTTGACTTGG-
tions 201, 203, and 307 [30]. The results of two GACT-3 0 and antisense, 5 0 -GTGGAACCCAATGCCTCTG-3 0 ;
representative cases are shown in Fig. 3A: samples the PCR products were digested with the restriction enzyme PstI.
210 M. Tanaka et al. / Legal Medicine 3 (2001) 205212
expected two fragments, indicating that their intron 2
was type c (data not shown).
4. Discussion
PCR assays for RHC/c and RHE/e genotyping have
been described [2326]. The RHC and RHc alleles
have been reported to differ by a single nucleotide
substitution in exon 1 (at position 48) and five base
changes in exon 2 (at positions 150, 178, 201, 203,
and 307) of the RHCE gene [14]. These six nucleotide
substitutions result in four amino acid substitutions.
One of the four substitutions occurs in exon 1, result-
ing in a Cys to Trp change at amino acid position 16.
The other three occur in exon 2 and involve amino
acid changes at residues 60 (Ile to Leu), 68 (Ser to
Asn), and 103 (Ser to Pro). Among these amino acids,
only the amino acid at residue 103 is predicted to be
located in the outer leaflet of the plasma membrane.
The polymorphism at position 307 for RHC/c seems
to be useful for genotyping of RHc, but not RHC,
because the sequence of the RHD gene in exon 2 is
identical to that of the RHC allele [14]. The difference
between the antigenicities of RHC and RHc has been
reported to be determined by the conversion of Trp at
residue 16 to Cys (C to G at the position 48 in exon 1)
and other unknown RhC-specific epitope(s) [32].
Here, we have developed a novel allele-specific
PCR method for RHC/c genotyping. We used this
method to examine 656 donor samples and found
that there was a correlation between RHC/c genotype
and serological phenotype in 639 samples, whereas
there was discrepancy in 17 cases who had a cc pheno-
type but a Cc genotype [30].
Cherif-Zahar et al. [29] analyzed the 5 0 flanking
sequence encompassing 600 bp upstream of the tran-
scription initiation site of the RHCE gene [29].
Although this sequence was obtained from an RhCcEe
clone, we found three nucleotide differences between
the RhC and Rhc 5 0 flanking region, and we showed
that one of these differences corresponded to position
48 of exon 1 in the RHCE gene.
In this study, for detection of the RHC/c allele, we
used RFLP to examine whether the sequence at 2292
(G/A) in the RHCE 5 0 flanking region was correlated
with the RhC/c phenotype. The allele-specific PCR
analysis revealed that exon 1 of the 17 exceptional
cases were all type Cc, which corresponded to the
genotype of the promoter region.
We sequenced the promoter region of the RHCE
gene of three ccEe and 14 ccee exceptional cases,
and found that the promoter regions were all Cc
type with no additional nucleotide substitution (data
not shown). Therefore, it is likely that the promoters
of these cases functioned normally, and consequently,
that the RhCE peptide was produced.
Poulter et al. [28] developed a PCR-RFLP method
for RHC/c genotyping on the basis of the DNA
sequence differences of intron 2 of the RHCE gene.
Using this assay, they had examined 105 unrelated
individuals from four different populations and
compared the results with those obtained by serologi-
cal testing of red cells, and they found a complete
correlation, but also pointed out the difficulty of asses-
sing D/c heteroduplex cases. When the conditions for
DNA amplification and PstI digestion were optimized,
the difference between the ratio of the RHCE and D
signals in Cde/cde compared with Cde/cDE samples
could be detected. We examined this method for our
samples. Unfortunately, RHC/c-specific fragments
could not be detected on agarose gels when small
volumes of PCR products were used (data not
shown). However, our 17 exceptional cases were clas-
sified as cc according to their method. The nucleotide
sequence of exon 2 isolated from our 17 exceptional
cases was identical to that of the normal RHc gene (data
not shown). These results suggest the possibility of
recombination between the RHC and RHc genes some-
where between exon 1 and intron 2 in these cases. It is
conceivable that this recombination results in the
expression of an unknown RhC-specific antigen.
We previously developed an allele-specific PCR for
RHC/c and RHE/e genotyping [30]. For detection of
the RHC allele, we designed primers on the basis of
the position 48 polymorphism (C/G) in exon 1 of the
RHCE gene. A complete correlation was observed in
the RHC/c genotyping of 656 samples between the
results obtained using this allele-specific PCR and
those obtained using our novel PCR-RFLP (Table
1). However, it is thought that the use of allele-speci-
fic primers to distinguish a one-nucleotide difference,
especially when it is a C/G-difference, decreases
primer efficiency [33]. Therefore, it is conceivable
that our newly developed PCR-RFLP may be more
reliable than the allele-specific PCR.
 M. Tanaka et al. / Legal Medicine 3 (2001) 205212
In conclusion, we found that the combination of
Poulters and our methods may prove to be more
accurate than either of the methods alone, and there-
fore useful for RHC/c genotyping.
Acknowledgements
The authors are sincerely grateful to M. Mizui, MT,
Hiroshima Red Cross Blood Center (Hiroshima,
Japan), and T. Taguchi, MT, Oita Red Cross Blood
Center (Oita, Japan) for providing the blood samples.
The authors would also like to thank M. Reid, Ph.D.,
New York Blood Center (New York, USA) for critical
and expert advice, and S. Taiko, MT, Murakami
Reference Laboratory (Tokyo, Japan) for serological
advice. The nucleotide sequence data reported in this
paper have been registered in the DDBJ/EMBL/
GenBank nucleotide sequence databases under the
accession numbers AB016499 and AB016502.
References
[1] Blanchard D, Bloy C, Hermand P, Cartoron JP, Saboori AM,
Smith BL, Agre P. Two-dimensional iodopeptide mapping
demonstrates erythrocyte RhD, c, and E polypeptides are
structurally homologous but non-identical. Blood
1988;72:14241427.
[2] Avent ND, Ridgwell K, Tanner MJ, Anstee DJ. cDNA cloning
of a 30 kDa erythrocyte membrane protein associated with Rh
(Rhesus)-blood-group-antigen expression. Biochem J
1990;271:821825.
[3] Mollison PL, Engelfriet CP, Contreras M. Blood transfusion
in clinical medicine. 10th ed.. Blackwell: Oxford, 1997.
[4] Gahmberg CG. Molecular identification of the human Rh0(D)
antigen. FEBS Lett 1982;140:9397.
[5] Moore S, Woodrow CF, McClelland DBL. Isolation of
membrane components associated with human red cell anti-
gens Rh(D), (c), (E) and Fya. Nature 1982;295:529531.
[6] Bloy C, Blanchard D, Lambin P, Goossens D, Rouger P,
Salmon C, Cartron JP. Human monoclonal antibody against
Rh(D) antigens: partial characterization of the Rh(D) polypep-
tide from human erythrocytes. Blood 1987;69:14911497.
[7] Avent ND, Ridgwell K, Mawby WJ. Protein-sequence studies
of Rh-related polypeptides suggest the presence of at least two
groups of proteins which associated in the human red-cell
membrane. Biochem J 1988(256):10431046.
[8] Saboori AM, Smith BL, Agre P. Polymorphism in the Mr
32,000 Rh protein purified from Rh(D) positive and negative
erythrocytes. Proc Natl Acad Sci USA 1988;85:40424045.
[9] Suyama K, Lunn R, Haller S, Goldtein J. Red cell surface
cystein residue (285) of D polypeptide is not essential for D
antigenicity. Transfusion 1995;35:653659.
211
[10] Cherif-Zahar B, Bloy C, Le Van Kim C, Blanchard D, Bailly
P, Hermond P, Salmon C, Cartron JP, Colin Y. Molecular
cloning and protein structure of a human blood group Rh
polypeptide. Proc Natl Acad Sci USA 1990;87:62436247.
[11] Le Van Kim C, Mouro I, Cherif-Zahar B, Raynal V, Cherrier
C, Cartron JP, Colin Y. Molecular cloning and primary struc-
ture of the human blood group RhD polypeptide. Proc Natl
Acad Sci USA 1992;89:1092511029.
[12] Kajii E, Umenishi F, Iwamoto S, Ikemoto S. Isolation of a new
cDNA clone encoding a Rh polypeptide associated with the
Rh blood group system. Hum Genet 1993;91:157162.
[13] Arce MA, Thompson ES, Wagner S, Cony KE, Ferdman BA,
Lubin DM. Molecular cloning of RhD cDNA derived from a
gene present in RhD-positive, but not RhD-negative indivi-
duals. Blood 1993;82:651655.
[14] Mouro I, Colin Y, Cherif-Zahar B, Cartron JP, Le Van Kim C.
Molecular genetic basis of the human Rhesus blood group
system. Nat Genet 1993;5:6265.
[15] Umenishi F, Kajii E, Ikemoto S. A new genetic polymorphism
of Rh polypeptide genes. Biochem J 1994;299:203206.
[16] Cartron JP. Defining the Rh blood group antigens. Biochem-
istry and molecular genetics. Blood Rev 1994;4:199212.
[17] Smythe JS, Avent ND, Judson PA, Parsons SF, Martin PG,
Anstee DJ. Expression of RHD and RHCE gene products
using retroviral transduction of K562 cells establishes the
molecular basis of Rh blood group antigens. Blood
1996;7:29682973.
[18] Kajii E. Advance in the Rh blood group system. Jpn J Legal
Med 1998;52:118.
[19] Okuda H, Kawano M, Iwamoto S, Tanaka M, Seno T, Okubo
Y, Kajii E. The RHD gene highly detectable in RhD-negative
Japanese donors. J Clin Invest 1997;100:373379.
[20] Bennett PR, Le Van Kim C, Colin Y, Warwick RM, Cherif-
Zahar B, Fisk NM, Cartron JP. Prenatal determination of fetal
RhD type by DNA amplification. N Engl J Med
1993;329:607610.
[21] Spence WC, Maddalena A, Demers DB, Bick DP. Molecular
analysis of the RhD genotype in fetuses at risk for RhD hemo-
lytic disease. Obstet Gynecol 1995;2:296298.
[22] Legler TJ, Eber SW, Lakomek M, Lynen R, Maas JH, Pekrun
A, Pekrun A, Repas-Humpe M, Schroter W, Kohler M. Appli-
cation of RHD and RHCE genotyping for correct blood group
determination in chronically transfused patients. Transfusion
1999;39:852855.
[23] Wolter LC, Hyland CA, Saul A. Refining the DNA poly-
morphism that associate with the Rhesus c phenotype.
Blood 1994;84:985991.
[24] Faas BHW, Simsek S, Bleeker PMM, Overbeeke NAM, Cuij-
pers HTHM, von dem Borne AEGKR, van der Schoot CE. Rh
E/e genotyping by allele-specific primer amplification. Blood
1995;85:829832.
[25] Le Van Kim C, Mouro I, Brossard Y, Chavinie J, Cartron JP,
Colin Y. PCR-based determination of Rhc and RhE status of
fetuses at risk of Rhc and RhE haemolytic disease. Br J
Haematol 1994;88:193195.
[26] Gassner C, Schmarda A, Kilga-Nogler S, Jenny-Feldkicher B,
Rainer E, Muller TH, Wagner FF, Flegel WA, Schonitzer D.
212 M. Tanaka et al. / Legal Medicine 3 (2001) 205212
RHD/CE typing by polymerase chain reaction using
sequence-specific primers. Transfusion 1997;37:10201026.
[27] Hyland CA, Wolter LC, Saul A, Saul A. Identification and
analysis of RH genes. Application of PCR and RFLP typing
tests. Transfus Med Rev 1995;4:289301.
[28] Poulter M, Kemp TJ, Carritt B. DNA-based Rhesus typing:
simultaneous determination of RHC and RHD status using the
polymerase chain reaction. Vox Sang 1996;70:164168.
[29] Cherif-Zahar B, Le Van Kim C, Rouillac C, Raynal V, Cartron
JP, Colin Y. Organization of the gene (RHCE) encoding the
human blood group RhCcEe antigens and characterization of
the promoter region. Genomics 1994;19:6874.
[30] Tanaka M, Seno T, Shibata H, Okubo Y, Okuda H, Kajii E,
Utsumi R. Genotyping for RhC/c and RhE/e by PCR using
allele-specific oligonucleotide primers. Jpn J Legal Med
1997;51:3238.
[31] Miller SA, Dykes DD, Polesky HF. A simple salting out
procedure for extracting DNA from human nucleotide cells.
Nucleic Acids Res 1988;16:1215.
[32] Noizat-Pirenne F, Mouro I, Le Pennec P. Evidence that serine
at position 103 is not sufficient for complete C antigen expres-
sion. Transfusion 1999;39(Suppl):103S.
[33] Lowe TMJ, Sharefkin J, Yang SQ, Dieffenbach CW. A
computer program for selection of oligonucleotide primers
for polymerase chain reaction. Nucleic Acids Res
1990;18:17571761.