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Annals of Clinical & Laboratory Science, vol. 35, no. 3, 2005
265
Identification of a Novel B Variant Allele at the ABO Locus in
Chinese Han Individuals with B Subgroup
Zhi-Hui Deng, Qiong Yu, Yan-Lian Lian, Guo-Guang Wu, Yu-Qing Su, and Xuan Zhang
Shen-Zhen Blood Center, Shen-Zhen Institute of Transfusion Medicine, Guang-Dong Province, China
Abstract. We studied the molecular genetic background of the B subgroup in the Chinese Han population
and identified a novel allele at the ABO locus. Ten control samples from randomly selected blood donors of
normal B phenotype and 6 samples from individuals diagnosed as B subgroup by serological tests were
genotyped by PCR-SSP and direct DNA sequencing at exons 6 and 7 of the ABO gene. Exons 6 and 7 and
the intervening intron 6 of B alleles from the 6 B subgroup samples were analyzed by cloning and haplotypesequencing. A novel B variant allele was identified in 2 individuals who were serologically-determined as
members of the Bx and Bw subgroups, respectively. The novel B allele differs from allele B101 by a single
695T>C missense mutation in exon 7. The family of the individual with Bx subgroup was studied; among
8 family members tested, 4 had the novel B variant allele. No mutation at exon 6 or 7 of the ABO gene was
detected in the 10 control samples or in the other 4 B subgroup samples. Mutation at position 695 where
T is replaced by C results in an amino acid change from Leu to Pro, which is predicted to diminish B
transferase activity. This indicates that alteration of the amino acid at position 232 is critical to the activity
of glycosyltransferases. (received 8 December 2004; accepted 5 February 2005)
Keywords: ABO blood group, B subgroup, cloning and DNA sequencing, novel B allele, glycosyltransferases
Introduction
The ABO blood group is beyond doubt the most
important blood group system in transplantation,
transfusion medicine, and paternity testing. It was
discovered by Landsteiner in 1901. In 1990, the
molecular backgrounds of the 3 main alleles A1, B,
and O, were identified by Yamamoto et al [1], who
cloned and sequenced the cDNA of the ABO gene.
In addition to the common ABO phenotypes, many
ABO subgroups with weak expression of the A or B
antigens on red blood cells (RBCs) have been found.
The subgroup phenotypes result from a variety of
molecular changes in the ABO allele [2]. Sequencing
the ABO alleles elucidates the molecular basis of the
ABO subgroups and helps to delineate the influence
Address correspondence to Dr. Zhi-Hui Deng, Shen-Zhen
Institute of Transfusion Medicine, Ni-Gang Xi Road, MeiGang Nan Street, Shen-Zhen, Guang-Dong Province, 518035,
People’s Republic of China; tel 86 755 8324 2567; fax 86 755
8322 1000; e-mail [email protected].
of amino acid variations on serologic specificity and
glycosyltransferase activity. Many ABO alleles have
been identified in various populations [3,4].
To determine the molecular basis of ABO
serologic subgroups in the Chinese Han population,
we have previously studied individuals with the A2
subgroup using cloning and sequence analysis. We
found that the A2 allele frequency and distribution
in the Chinese Han population are quite different
from other populations. A2-5 is the predominant
allele of A2 in the Chinese Han population [5]. We
have also identified a novel O1 variant allele in an
A2 subgroup individual (genotyped as A2O1). This
novel O 1 allele (GenBank accession number:
AY374123) is identical to the ABO*O101 allele
except for A-deletion at position 496 in exon 7 [6].
In this study, we report a novel B allele,
identified in two B-subgroup individuals, which
differs from the allele B101 by single 695T>C
missense mutation in exon 7. A family study
demonstrated that this novel allele is inheritable and
0091-7370/05/0300-0265. $1.25. © 2005 by the Association of Clinical Scientists, Inc.
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266 Annals of Clinical & Laboratory Science, vol. 35, no. 3, 2005
rules out a possible artifact or somatic mutation as
the cause of nucleotide (nt)695T>C.
Materials and Methods
Samples. Ten randomly-selected blood samples from blood
donors with common B phenotype were used as controls. Six
B subtype samples, which had previously been serologically
typed, were obtained from our blood group reference laboratory. The blood samples all came from healthy blood donors
of the Chinese Han population. Additionally, blood samples
were obtained from 8 relatives of a Bx subgroup donor; these
family members were all recruited with informed consent.
Venous blood samples (3 ml) were drawn into EDTA tubes.
Serological tests. Routine serological testing (eg, RBC and serum
blood grouping procedures; adsorption and elution with antiB and A, B, and H substance by saliva test) was performed by
standard serological methods. The antisera included monoclonal anti-A and anti-B (Ortho, Raritan, NJ, USA), polyclonal
anti-B (blend of human serum), and monoclonal anti-A,B
(Immucor, Norcross, GA, USA). Lectins from Ulex europaeus
were used for anti-H (Dominion, Nova Scotia, Canada).
Genotyping by PCR-SSP. Four pairs of sequence-specific
primers (SSP) were synthesized. The first primer pair, designed
by us (1sense/2antisense):
5’-gga agg atg tcc tcg tgg tg-3’; 5’-tga gga tgt gga tgt tga at-3’)
was used to amply the common A allele. Three other pairs,
designed by Seltsam [7], were (27sense /33 antisense primers):
5’-cat tgt ctg gga ggg cac g-3’; 5’-ctt gat ggc aaa cac agt taa c-3’;
(21sense/33 antisense primers):
5’-gga agg atg tcc tcg tgg ta-3’; 5’-ctt gat ggc aaa cac agt tac c-3’;
(41sense/46 antisense primers):
5’-acg tgg ctt tcc tga agc tg-3’; 5’-gcg ggg cac cgc ggc ca-3’)
were used to amplify the common B, O1, and A102 alleles
respectively.
Polymerase chain reaction (PCR) amplification was
carried out in a reaction volume of 10 µl containing 1 x PCR
buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM
MgCl2), primer mix, 80 µM of each dNTP, 1U Taq polymerase,
and 100 ng of genomic DNA. The reaction was performed in
a GeneAmp PCR system 9700 using the following PCR cycling
parameters: 1 cycle at 95°C for 5 min; 30 cycles at 95°C for 30
sec, 60°C for 30 sec, and 72°C for 90 sec; followed by a final
extension for 72°C for 5 min. PCR products were
electrophoresed on 4% agarose gels containing 0.5 ug/ml
ethidium bromide and visualized by UV transillumination.
Direct sequencing of exons 6 and 7 of ABO gene. Because
91% of the ABO coding sequences lie in exons 6 and 7 [8],
PCR-based gene analyses were performed on the 2 exons.
Primer pairs mol-46/mol-57 and mol-71/mol-101 described
in a previous study [9] were used to amplify exons 6 and 7.
The PCR fragment sizes for exons 6 and 7 were 252 bp (251
bp for O1) and 843 bp, respectively. PCR amplification was
carried out in a reaction volume of 50 µl containing 1 x PCR
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266
Fig. 1. Pedigree and RBC phenotypes of the family members of
a Bx subgroup donor (arrow).
buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM
MgCl2 ), 400 µM each of dNTP, 0.1µM of each primer pair,
300-500 ng of genomic DNA, and 2.5 U of Taq DNA polymerase. Amplification was carried out under the following
conditions: 95°C for 10 min; 10 cycles: 94°C for 60 sec, 63°C
for 90 sec, and 72°C for 60 sec; 25 cycles: 94°C for 60 sec,
61°C for 90 sec, and 72°C for 60 sec; followed by a final
elongation at 72°C for 10 min.The PCR products were purified
using Takara DNA Fragment Purification Kit (TaKaRa, Dalian,
China) according to the manufacturer’s instruction. The
purified PCR products were directly sequenced using BigDye
Terminator Cycle Sequencing Ready Reaction Kit (Applied
Biosystems) and analyzed by an ABI PRISM 3100 Genetic
Analyzer (Applied Biosystems).
Cloning and haplotype sequencing of exon 6 , intron 6, and
exon 7 at the ABO locus. To determine the haploid type of
ABO gene, a fragment of 2170 bp spanning exon 6, intron 6,
and exon 7 was amplified using the following primer pair,
designed by us:
5’-cgt gaa ggg tgg tca gag ga -3’; 5’-gtt act cac aac agg acg gac -3’.
The PCR amplification was carried out in a volume of 50 µl
containing 2 µl of 2 x GC buffer I/II,100 µM each of dNTPs,
0.1 µM each of 2 primers, 300 to 500 ng of genomic DNA,
and 2 U of LA Taq polymerase (Takara). The reaction was
performed in a GeneAmp PCR system 9600 using the following
PCR conditions: 1 cycle of 95°C for 10 min; 30 cycles of 94°C
for 30 sec, 60°C for 30 sec, and 72°C for 150 sec; and finally
72°C for 10 min. The gel-purified PCR product was cloned
into the pCRII vector using TOPO cloning kit (Invitrogen,
Groningen, Netherlands). Isolated clones containing the B allele
were screened using the 27s/33as primer pair by the PCR-SSP
method. The haplotype sequencing reaction was performed in
a final volume of 10 µl using the following 5 forward sequencing
primers:
Name
AF1
AF2
AF3
AF4
AF5
Location
Intron 5
Intron 6
Intron 6
Exon 7
Exon 7
Sequences
5’-GGC GGC CGT GTG CCA GA-3’
5’-TTG TCC TCC CAG AGG GTA GA-3’
5’-CAA CCG CAG ACA CAT ACT TGA-3’
5’-CAG GAC GGG CCT CCT GCA-3’
5’-CCA GTC CCA GGC CTA CAT-3’.
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Novel B variant allele in Chinese Han subjects with B subgroup 267
Results
Serologic phenotypes and respective genotypes. The
serologic phenotypes and genotypes are shown in
Table 1. Six B-subgroup samples (Samples 1 to 6)
were typed as Bw, ABel, Bm, Bx, Bw, and B3 by
adsorption-elution and saliva testing, and were also
genotyped by PCR-SSP. Consistent with the
serologic phenotypes, the genotypes were all B1O1
except sample 2, which was typed as A102B. A family
study was performed in 7 relatives of a Bx subgroup
individual (Fig. 1). Of the 8 family members, 4
(including the propositus, her mother, uncle, and
sister) were identified as Bx by serologic testing. Both
grandparents of the propositus had died, so they were
unavailable for testing
Directing sequencing and cloning analysis. In this
study, each B allelic clone was selected for haplotype
sequencing analysis, which allowed us to identify
the nucleotide sequence of B allele at exons 6 and 7
for all of the B subgroup samples. According to each
sample’s direct sequencing results, which were the
heterozygous and haplotype sequence of B allele, we
deduced the nucleotide sequence of another ABO
haplotype belonging to the A allele or O allele. We
defined each ABO allele using the method described
by Chester and Olsson [10]. As a result, 4 common
B samples were defined as B101O1; the other 6
Fig. 2. Sequencing result at position 695 in wild-type B allele.
The 2 nucleotides indicated by dashes are G and C, respectively.
common B samples were B101O1 v ; no novel
mutation was found at exons 6 or 7 in the control
group. For the 6 B-subgroup samples, sample 1 to 3
and sample 6 were defined as B101O1v, A102B101,
B101O1, and B101O1, respectively. The single
nucleotide polymorphism (SNP) sites of sample 4
Table 1. Findings in 6 B subgroup samples and 10 control samples detected by serological tests and PCR-SSP.
Samples
sample 1
sample 2
sample 3
sample 4
sample 5
sample 6
control
group*
Anti-A Anti-B Anti-AB Anti-H
Ac
Bc
Oc
Absorption
& elution test
Saliva
test
Phenotype Genotype
3+
-
w+
w+
mf
4+
w+
w+
mf
3+
2+
3+
4+
4+
4+
2+
4+
3+
+
3+
w+
-
-
no B antigen
B antigen
B antigen
B antigen
no B antigen
B antigen
B, H
A, H
B, H
B, H
B, H
B, H
Bw
ABel
Bm
Bx
Bw
B3
BO1
A102B
B1O1
B1O1
B1O1
B1O1
-
4+
4+
4+
4+
-
-
-
B, H
B
B1O1
* The control group comprised 10 samples of common B phenotype, randomly selected from blood donors.
Abbreviations: Ac = A type RBC blend; Bc = B type RBC blend; Oc = O type RBC blend.
w+ = weak; - = negative; mf = mixed-field hemagglutination of RBCs.
+, 2+, 3+, 4+ = degrees of hemagglutination of RBC.
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268 Annals of Clinical & Laboratory Science, vol. 35, no. 3, 2005
Fig. 3. Sequencing result at position 695 in novel
B variant allele.
at one haplotype were determined as 261G deletion,
297A, 467C, 526C, 657C, 695T, 703G, 796C,
803G, 930G, and 1096G; the sample can be defined
as wild-type O101. However, other haplotype SNP
sites of sample 4 were determined as 261G, 297G,
467C, 526G, 657T, 695C, 703A, 797A, 803C,
930A, 1096A, and a novel SNP site mutation at
(nt)695 (695T>C) in exon 7 was identified; we
define this as a novel variant B allele. The family
study showed that this novel B allele is present in 4
family members. The finding that this novel B allele
is inheritable rules out a possible artifact or somatic
mutation as the cause of the (nt)695T>C missense
(Figs. 2,3). The sequencing results of sample 5 (Bw
subgroup) were identical to sample 4 and the
(nt)695T>C mutation was also determined as a B
allelic haplotype.
Discussion
The weak B phenotype is more frequent than the
weak A subgroup in the Chinese Han population.
The subgroup B is characterized by no agglutination,
or only weak hemagglutination, of RBCs with antiB and anti-A-anti-B antibody. Without excellent
operating technique and a proper titer of antiserum,
the weak B phenotype is often misrecognized as O
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phenotype. Although the B antigen expression of
weak B individuals is very weak, its clinical effect is
clear. In this report, we identified the molecular basis
of the B subgroup polymorphism. Based on
serological findings, the B subgroups were classified
into 5 categories [11]: B3, Bx, Bm, Bel, and Bw. We
adopt the designation Bw for this antigen for those
B variants that do not fit in the other 4 categories
[12].
Until now, many novel ABO alleles have been
identified and characterized. According to the Blood
Group Antigen Gene Mutation Database [13], more
than 20 B alleles have been previously reported and
have been classified in 6 categories: B1, B3, Bel, Bw,
CisAB, and B(A). Compared to the reference A101
and B101 allelic sequence, the B variant alleles have
3 highly-conserved mutations at nt703, nt796, and
nt803; the other mutation sites vary from B allele
to B allele. Take alleles of B3, Bel, and Bw subgroups
for instance: mutations at nt703, nt796, and nt803
are the same for all these alleles, and one or more of
an additional 13 new and rare nucleotide variations
(eg, C502T, G539A, A548G, T641G, G669T,
C721T, T863G, G871A, C873G, C1036G,
A1037T, C1054T, and G1055A) are also observed.
Molecular cloning analysis of the ABO gene
allows the elucidation of the molecular basis of its
various alleles. Lin et al [14] identified a unique
502C>T mutation in a Bel-subgroup individual in
Taiwan; Lung-Chih Yu et al [15] found a 247G>T
mutation in a B3 subgroup individual; Estalote et al
[16] found a novel 556A>G missense mutation at
exon 7 in a Caucasian family.
In the present study, we singled out B allelic
clones and performed sequencing analysis at exons
6 and 7 and the interval intron 6. A novel B allele
was found in 2 individuals who were serologicallyidentified as Bx and Bw, respectively. The novel B
allele shared high sequence homology with B101
and differed from B101 by single 695T>C missense
mutation in exon 7. We define it as a new variant
allele. To confirm our findings, we obtained blood
samples from relatives of the Bx propositus. The
family study showed that 4 of 8 relatives carry this
novel B allele. These findings indicate that this novel
B allele is inheritable and rule out a possible artifact
or somatic mutation.
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Novel B variant allele in Chinese Han subjects with B subgroup
Among the 6 B-subgroup samples, 2 that were
serologically-determined as Bx or Bw were found to
carry the novel B variant allele. This suggests that
the novel B allele may be frequent in the Chinese
Han population, and indicates that different
serologic subgroups may have a common molecular
basis. Monoclonal reagents have been replacing
traditional polyclonal antisera and may have been
causing difficulties in serological classification of the
subgroups. The novel mutation, which changes T
to C at nt695 in exon 7, results in an amino acid
change of Leu to Pro. The mutation hypothetically
diminishes the α -1,3-galactosyltransferase (B
transferase) activity that is responsible for the
formation of the B determinant by transfer of Gal
from the UDP-Gal donor to the H precursor. In a
normal adult, B antigen sites number about 600,000
to 800,000. While we observed that RBCs from the
2 individuals reacted weakly with anti-B sera, we
think that some B subgroups may have a quantitative
difference in their B antigens. We predict that the
nucleotide at position 695 is critical for glycosyltransferase activity.
Although no new mutation was identified in
the 4 other B-subgroup individuals, further study
needs to be undertaken as to whether additional
mutations exist in other exons or introns, or in the
promoter region of the B subgroup gene, which
could contribute to the B subgroup phenotype.
In summary, this study documents a novel B
allele in the ABO blood group. Our findings suggest
that the molecular determinants of the B subgroup
in the Chinese Han population may differ from
those reported in other populations.
Acknowledgments.
The authors thank the Wu family for providing
blood and DNA samples. This work was supported
by the Research Fund of Guang-Dong Health
Department (Project 2001641) and by the Research
Fund of ShenZhen Bureau of Science & Technology
(Project 200304217).
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