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Molecular Genetic Basis of ABO Blood Group System
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DOI: 10.13140/RG.2.2.23407.79527
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ABO Blood Groups
Molecular Genetic Basis of ABO Blood Group System
This website was created by Fumiichiro Yamamoto, Ph.D., to disseminate knowledge on the
molecular genetic basis of the ABO system.
Karl Landsteiner discovered the ABO system in 1900, distinguishing it as one of the most important
blood group systems in transfusion medicine. The system consists of A and B antigens and their
corresponding antibodies. The underlying factor differentiating the ABO system from others, such as
the Rh system, is the presence of antibodies against A and B antigens. These antibodies are present
in individuals who do not express A and B antigens, and cause the first mismatched blood transfusion
to be possibly fatal. The discovery of the ABO blood group system paved the way for safe blood
transfusion.
Due to its complexity, exploration of the ABO system peaks interest not only in transfusion medicine,
but also in a variety of scientific fields. In addition to the four major groups (A, B, AB, O), we know of
more than a dozen existing subgroups that exhibit different patterns and degrees of agglutination.
Additionally, A and B antigens are found not only on red blood cells (RBCs) but also on the surface of
other cell types and in secretions. As such, the system is often referred to as the “histo-blood group
system.” The presence of A and B antigens on cells other than RBCs emphasizes the importance of
ABO blood type matching not only in blood transfusions, but also in cell, tissue, and organ
transplantations.
Both the synthesis and properties of A and B antigens raises many important questions on their roles
not only in medicine but also in many aspects of biology. A and B antigens are synthesized by a series
of enzymatic reactions catalyzed by enzymes called glycosyltransferases. In fact, the final step in
producing these antigens requires a glycosyltransferase, which is encoded by the functional A and B
alleles at the ABO genetic locus. The fact that allele frequencies vary amongst different races raises
interesting questions on the relevance of ABO blood type on population studies, anthropology and
human genetics. Another interesting characteristic of A and B antigens is their presence in animals
other than human beings. The glycosyltransferases involved in A/B antigen production in humans
also exhibit the same enzymatic purposes in animals. Therefore, the ABO blood group system is also
of evolutionary and enzymatic significance. A/B antigens also exhibit dynamic changes during
development and pathogenesis, suggesting their importance in cancer, molecular, cellular and
developmental biology.
Safer blood transfusion, conceived by Landsteiner and improved upon by many others, primarily
immunohematologists, has become a routine medical practice. Since our cloning of the ABO gene in
1990, progress has been made in the structural and functional analyses of ABO genes and A/B
transferases at the molecular level. I hope that readers find these web pages interesting and useful,
and that they both help facilitate a better understanding of the scientific bases of the ABO system,
oligosaccharide ABH antigens, A and B transferases, and ABO genes, and aid in applying this
information to clinical applications.
02. Immunogenetics of the Histo-blood group ABO System
A slide presentation Dr. Yamamoto gave at Osaka University was modified to prepare the following
web pages. The most up-to-date unpublished information has been omitted. Nonetheless, you may
still learn the basics of ABO genes, A and B transferases, and ABH antigens from this material. You
may also obtain a clear view of both what has been answered and what remains to be achieved
scientifically in the study of the ABO system. If you would like a copy of the PowerPoint file of this
presentation
for
use
in
your
fyamamoto@carrerasresearch.org
curriculum,
etc.,
please
contact
Dr.
Yamamoto
at
03. Discovery of ABO Blood Group System
Fumiichiro Yamamoto is the Senior Group Leader at the Institute of Predicative and Personalized
Medicine of Cancer (IMPPC) in Barcelona, Spain. For his elucidation of the molecular genetic basis
of the ABO system, Dr. Yamamoto has received international recognition, including the Jean Julliard
Prize from the International Society of Blood Transfusion.
The ABO system is one of the most important blood group systems in the safe practice of blood
transfusion. Karl Landsteiner discovered the ABO blood group in 1900 when he separated cellular
and liquid components from both his and his colleagues’ blood mixed in combination. As indicated by
the negative symbol (-) in the table above, no agglutination of red blood cells was observed when
both components from the same individuals were mixed. However, RBC agglutination was observed
in some combinations, as indicated with the positive symbol (+). From the results gleaned from his
studies, Landsteiner realized that people could be grouped depending on the agglutination pattern of
their RBCs. For example, Dr. St. and Mr. Land. belong to one group. Similarly, Dr. Plee. and Mr. Zar.
belong to a disparate second group, and Dr. Sturl. and Dr. Erdh belong to a third group. In the next
year, a fourth group was discovered by Sturle and von Decastello, Landsteiner's colleagues, and
these four groups became what is now known as the ABO group system (A, B, AB, and O groups).
However, the most important finding obtained from these experiments is that only ABO-matched
blood that does not cause RBC agglutination should be used for transfusion.
In order to explain the RBC agglutination phenomenon, Landsteiner postulated that 2 different
antigens (A and B) are found on the surface of RBCs, and the "naturally occurring" antibodies against
these antigens are found in the plasma of individuals who do not express them (The Landsteiner’s
Law). For example, blood group A individuals express the A antigen on their RBCs and possess antiB antibodies in their plasma. Similarly, blood group B individuals express the B antigen on their RBCs
and possess anti-A antibodies in their plasma. Blood group AB individuals express both A and B
antigens on their RBCs and possess neither anti-A nor anti-B antibodies, whereas blood group O
individuals express neither A nor B antigens on their RBCs but possess both anti-A and anti-B
antibodies.
ABO blood groups are hereditary, and their mode of inheritance was explained by Bernstein’s one
gene-three allelic model. In this model, Bernstein postulated that there are three alleles, A, B, and O,
at a single ABO genetic locus, and that A and B alleles are co-dominant against the recessive O allele.
This produces 6 genotypes (AA, AO, BB, BO, AB, and OO) resulting in 4 phenotypes.
The allele frequencies of A, B, and O vary among different races. For example, before the prevalence
of intermarriage with other races, American Indians were all type O. Therefore, the ABO blood group
system is also a subject of interest in population genetics and anthropology.
04. A and B Antigens
A and B antigens were originally identified on red blood cells. However, they were also later identified
on other types of cells and in secretion. For example, endothelial cells that form the linings of
capillaries express these antigens depending on blood type. Therefore, the ABO blood group is
important not only for blood transfusion, but also for cell/tissue/organ transplantation. Also, blood,
hairs, and seminal fluid are important pieces of evidence at crime scenes. Therefore, ABO blood
typing has played an important role in excluding suspects in forensic investigations. Additionally, A
and B antigens are not solely restricted to humans. Both the same and similar antigens have been
found in other species of organisms. For example, chimpanzees express A and O blood groups,
whereas gorillas express B blood groups. In addition to primates, many mammals and vertebrates,
plants, and certain microorganisms have been shown to express the same or similar antigens. The
evolution of the ABO system is, therefore, of scientific interest.
The expression of A and B antigens is not always constant. It fluctuates during development,
differentiation, and even carcinogenesis of cells. The elucidation of how the expression of these
antigens is controlled was an important issue to research. Additionally, why "naturally occurring"
antibodies against A and B antigens are present in the plasma of individuals who do not express
those antigens is an important question to be answered.
A and B antigens are not protein antigens but rather oligosaccharide antigens with the chemical
structures of GalNAc α1-3 (Fuc α1-2) Gal- and Gal α1-3 (Fuc α1-2) Gal-, respectively. The
immunodominant sugars are GalNAc (N-acetyl-D-galactosamine) and D-galactose for A and B
antigens, respectively. The difference between these two sugars is that GalNAc has -NHCOCH3 at
the C2 position whereas galactose has a smaller –OH at this position.
05. A and B transferases
Once the chemical structures defining the ABO blood system were determined, Watkins and Morgan,
and separately Ceppellini, proposed a hypothesis on the biosynthetic pathway of these antigens. They
postulated that A and B antigens are produced from the same precursor as an H antigen, which is
abundant in individuals with blood group O. A and B antigens are formed by the action of
glycosyltransferases encoded by functional alleles at the ABO genetic locus (The Central Dogma of
ABO). Namely, the A allele encodes A transferase, which transfers a GalNAc to the H antigen. This
synthesizes the A antigen. Similarly, the B allele encodes B transferase, which transfers a galactose
molecule to the H antigen to synthesize the B antigen.
In addition to A and B transferases, there are other glycosyltransferases with α1-3 Gal(NAc)
transferase activity/specificity. These include α1-3 Gal transferase to synthesize α1-3 Gal epitope
(Gal α1-3 Gal β1-4 GlcNAc-), isogloboside 3 synthase to synthesize iGb3 ceramide (Gal α1-3 Gal β14 GlcCer-), and Forssman glycolipid synthase to synthesize the Forssman antigen (GalNAc α1-3
GalNAc β1-3 Gal α1-4 Gal β1-4 GlcCer-). Whether these glycosyltransferase genes are evolutionarily
related to ABO genes was an important question.
06. A transferase cDNA cloning
In 1990, we cloned cDNAs encoding human A transferase (Yamamoto et al., 1990) based on the
partial amino acid sequence of the isolated protein (Clausen et al., 1990). We reverse-translated the
amino acid sequences of the tryptic peptides from the isolated protein into nucleotide sequences.
Based on the sequence information, we prepared 3 degenerate oligonucleotides, 2 of which were
later used as PCR primers. The remaining oligonucleotide for the internal sequence was used as a
probe. After RT-PCR of RNA prepared from human stomach cancer MKN45 cell line cells, which are
known to express large amounts of A antigens and a high activity of A transferase, the PCR products
were electrophoresed through an agarose gel. The DNA was denatured, neutralized, and Southern
transferred onto a nitrocellulose membrane. It was then fixed and subsequently hybridized with the
32P-radiolabelled oligonucleotide probe for the internal sequence. Although the PCR products
exhibited smears of bands in the agarose gel, a single band of the DNA fragment of the expected
band size of 98 base pairs (bps) was hybridized. As such, the band was cut, PCR-amplified, and
cloned into a plasmid vector. After transformation of E. coli, bacterial clones that contained the
plasmid with a correct insert were identified. The 98 bp DNA fragment was then used as a probe to
screen the MKN45 cDNA library prepared in the lambda 10 phage vector.
Clausen, H., White, T., Takio, K., Titani, K., Stroud, M., Holmes, E., Karkov, J., Thim, L., and Hakomori,
S. (1990). Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc
alpha 1->2Gal alpha 1->3-N-acetylgalactosaminyltransferase from human lung tissue. J Biol Chem
265, 1139-1145. (http://www.jbc.org/cgi/reprint/265/2/1139)
Yamamoto, F., Marken, J., Tsuji, T., White, T., Clausen, H., and Hakomori, S. (1990). Cloning and
characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1-2Gal alpha 1-3GalNAc
transferase
(histo-blood
group
A
transferase)
(http://www.jbc.org/cgi/reprint/265/2/1146)
mRNA.
J
Biol
Chem
265,
1146-1151.
07. Northern Hybridization Results
Once cDNA clones encoding potential A transferase were identified, we performed Northern
hybridization experiments using RNAs prepared from cell line cells that exhibited different ABO
phenotypes. The results showed that homologous messages were present in cell line cells with
different ABO phenotypes. Therefore, we attempted to isolate cDNA clones containing homologous
sequences from those cell line cells. (The ABO phenotypes of the cell line cells and the blood types
of the patients from whom the cell lines were derived are shown in the parentheses. For example,
MKN45 cells express A antigens, but the blood type of the patient was unknown, while SW48 cells
express A and B antigens and were derived from a blood type AB patient).
Yamamoto, F., Marken, J., Tsuji, T., White, T., Clausen, H., and Hakomori, S. (1990). Cloning and
characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1-2Gal alpha 1-3GalNAc
transferase
(histo-blood
group
A
transferase)
(http://www.jbc.org/cgi/reprint/265/2/1146)
mRNA.
J
Biol
Chem
265,
1146-1151.
08. A/B/O Allelic cDNAs
We initially prepared two additional cDNA libraries using RNAs from human colon carcinoma SW948
and SW48 cell line cells (Yamamoto, 1990). The SW48 cell line was derived from a patient with AB
blood type, whereas the SW948 cell line was derived from a different patient with O blood type. We
used a probe prepared from the FY59-5 cDNA clone that was isolated from the MKN45 cDNA library
and contained a cDNA fragment much larger than 98 bps. We identified clones that were hybridized
with the probe, and determined the nucleotide sequences. The cDNA clones from the SW48 cDNA
library were divided into 2 groups depending on sequence difference; one group showed more
homology to the cDNA sequence of the MKN45 cDNA library than the other group. Therefore, that
group was assumed to represent the A allele, whereas the group with more differences was assumed
to represent the B allele. Conversely, the cDNA clones from the SW948 cDNA library showed identical
nucleotide sequences, though several clones showed different splicing patterns. We realized that,
compared with the cDNA clones from the MKN45 cDNA library, the cDNA clones from SW948 had a
single nucleotide deletion. Because a single nucleotide in the coding region may change the frame of
codons and produce non-functional proteins due to a frameshift, we assumed that those cDNAs
represented the O allele.
We then constructed 2 additional cDNA libraries of human colon adenocarcinoma (cell lines
COLO205 and SW1417). The COLO205 cell line cells exhibited the O phenotype, though the ABO
blood group of the patient was not known. The SW1417 cell line cells were derived from a patient with
B blood type and exhibited the B phenotype. The screening of these two cDNA libraries identified
dozens of cDNA clones that contained homologous sequences. DNA sequencing was performed,
and we determined that the cDNA clones from COLO205 showed an identical nucleotide sequence,
though there were variations in the splicing pattern. All of the cDNA clones contained the single
nucleotide deletion previously identified in the SW948 cDNA clones. Although the cDNA clones also
possessed additional nucleotide substitutions, they were assumed to represent the O allele because
of the presence of the single nucleotide deletion. The cDNA clones from SW1417 were divided into 2
groups by differences in the nucleotide sequence. One group showed the same sequence as the
cDNAs from SW948, which was assumed to represent the O allele, and other group showed the same
sequence as the cDNAs from SW48, which was assumed to represent the B allele. This demonstrated
that the blood type B patient from whom the SW1417 cell line cells derived had a BO genotype and
not a BB genotype. This experiment became the first successful example of ABO genotyping. Before
this discovery, it was impossible to determine the ABO genotype of individuals with blood type A or B
by immunological methods.
Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990). Molecular genetic basis
of
the
histo-blood
group
ABO
system.
(http://www.nature.com/nature/journal/v345/n6272/abs/)
Nature
345,
229-233.
09. Deduced Amino Acid Sequences of A/B/O Alleles
Once nucleotide sequences were determined for the three major alleles of A (A1), B, and O, we
deduced the amino acid sequences of their coding regions. We found that O alleles encode truncated
proteins due to the single nucleotide deletion (261delG) described above. This explained the nonfunctionality of O alleles. We also found that there are several nucleotide substitutions between A and
B alleles - 4 of which result in amino acid substitutions. Those are arginine (R), glycine (G), leucine
(L), and glycine (G) at codons 176, 235, 266, and 268 in A transferase, and glycine (G), serine (S),
methionine (M), and alanine (A) at the same codons in B transferase.
Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990). Molecular genetic basis
of
the
histo-blood
group
ABO
system.
(http://www.nature.com/nature/journal/v345/n6272/abs/)
Nature
345,
229-233.
10. Restriction Fragment Length Polymorphism (A/B Allele vs. O Allele)
Once these important differences were identified, we observed restriction enzyme cleavage sites at
several of the differentiated sites. For example, the sequence containing the O allele-specific single
nucleotide deletion can be cleaved with KpnI, whereas the sequence without the deletion can be
cleaved with BstEII. In this Southern hybridization experiment, genomic DNA from the cell line cells
that were used to prepare the cDNA library was cleaved with either BstEII or KpnI, electrophoresed,
Southern transferred, and hybridized with the radio-labeled A transferase probe. MKN45 genomic
DNA was cleaved with BstEII but not with KpnI. Conversely, half of the SW1417 genomic DNA was
cleaved with BstEII and the remaining half was cleaved with KpnI, showing that this cell line is
heterozygous.
Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990). Molecular genetic basis
of
the
histo-blood
group
ABO
system.
(http://www.nature.com/nature/journal/v345/n6272/abs/)
Nature
345,
229-233.
11. RFLP (A/O Alleles vs. B Allele)
Similarly, the sequences around the first and second of the four amino acid substitutions that
discriminate A/O and B alleles can be cleaved by BssHII and NarI, and HpaII and AluI, respectively.
Using restriction fragment length polymorphism (RFLP), we confirmed the presence of these
differences in genomic DNAs from the cell lines used to prepare cDNA libraries (these distinctions in
RNAs were previously determined by the nucleotide sequencing of cloned cDNAs). As opposed
to KpnI and BstEII, which recognize and cleave longer nucleotide sequences, HpaII and AluI are
frequent cutters, recognizing and cleaving shorter sequences. As such, we used PCR to amplify DNA
fragments containing those restriction sites and then digested with these enzymes, rather than
Southern hybridization. We used the same strategy for the BssHII and NarI distinction, though they
are not frequent cutters because of easier experimental protocols. When we performed these
experiments, the specificity of the Taq DNA polymerase was not as good as it is now. Therefore,
several additional bands are visible in the background. Nonetheless, the presence/absence of the
restriction sites could be determined easily.
Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990). Molecular genetic basis
of
the
histo-blood
group
ABO
system.
(http://www.nature.com/nature/journal/v345/n6272/abs/)
Nature
345,
229-233.
12. ABO Genotyping of Blood Specimens
In order to examine whether the identified changes were common or uncommon, we next examined
the RFLP of genomic DNAs prepared from blood specimens with known ABO types. We analyzed 14
specimens with a variety of ABO types. Based on the RFLP results, we deduced the ABO genotypes
of the individual specimens and later compared them with the ABO types of the specimens. We did
not find any contradictions, suggesting that those differences in the nucleotide sequences are
commonly present.
Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990). Molecular genetic basis
of
the
histo-blood
group
ABO
system.
(http://www.nature.com/nature/journal/v345/n6272/abs/)
Nature
345,
229-233.
13. ABO Alleles (A and B Alleles)
Again, there are several nucleotide substitutions between A and B alleles - 4 of which result in amino
acid substitutions. Those are arginine (R), glycine (G), leucine (L), and glycine (G) at codons 176,
235, 266, and 268 in A transferase, and glycine (G), serine (S), methionine (M), and alanine (A) at
those codons in B transferase.
Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990). Molecular genetic basis
of
the
histo-blood
group
ABO
system.
(http://www.nature.com/nature/journal/v345/n6272/abs/)
Nature
345,
229-233.
14. ABO Alleles (O Alleles)
We chose one of the A1 alleles that we identified as a standard and named this allele the A101 allele.
The terminology was based on the phenotype plus a 2-digit number in the order of discovery,
assuming that less than 100 alleles would be found for each individual allelotype. During the cloning
of the ABO allelic cDNAs, we identified 2 kinds of O allelic cDNAs. One type has a single nucleotide
deletion at nucleotide 261 when compared to A101, which was later named O01. Another type of O
allelic cDNA contained several nucleotide substitutions in addition to the single nucleotide deletion.
We later named this O allele O02. During the characterization of subgroup alleles, we also
encountered another type of O allele that lacked the single nucleotide deletion. We found that this
allele (O03) contained 2 amino acid substitutions (R176G and G268R), which seemed to abolish A
transferase activity of the protein encoded by the allele (Yamamoto et al., 1993).
Yamamoto, F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990). Molecular genetic basis
of
the
histo-blood
group
ABO
system.
(http://www.nature.com/nature/journal/v345/n6272/abs/)
Nature
345,
229-233.
Yamamoto, F., McNeill, P.D., Yamamoto, M., Hakomori, S., Bromilow, I.M., and Duguid, J.K. (1993b).
Molecular genetic analysis of the ABO blood group system: 4. Another type of O allele. Vox Sang 64,
175-178.
15. ABO Alleles (A and B Subgroup Alleles)
In addition to the 4 major groups of A1, B (B1), A1B, and O, there are additional ABO subgroups.
The classification of these subgroups is based on differences in the degree (strength) of agglutination
of RBCs with anti-A, anti-B, and anti-A,B reagents, the presence of anti-A, anti-B, and anti-A,B
antibodies in sera, and the secretion of A and B antigens in saliva, among others. The weak
subgroups include A2, A3, Ax, Ael, Aint, Am, Aw, Ax, B3, Bel, Bw, and Bx. By 1993, we extended our
study of the molecular genetic basis of the ABO system to several of the A and B subgroups.
Compared with A101, the A201 allele that specified the A2 phenotype possessed 2 differences. The
first resulted in an amino acid substitution (proline to leucine) at codon 156 (P156L). The other
produced a single nucleotide deletion at nucleotide 1060 (1060delC), which caused frame-shifting
and resulted in a protein with an additional 20 amino acid residues at the C-terminus (Yamamoto et
al., 1992). The A301 allele that specified the A3 phenotype had a single nucleotide substitution that
resulted in an amino acid substitution from aspartic acid to asparagine (D291N). Additionally, the
Ax01 allele that specified the Ax phenotype had a single nucleotide substitution that resulted in an
amino acid substitution of phenylalanine to isoleucine (F216I) (Yamamoto et al., 1993a; Yamamoto
et al., 1993c). The B301 allele was identical to B101 except for a single nucleotide substitution
resulting in an amino acid substitution from arginine to tryptophan (R352W) (Yamamoto et al., 1993a).
Yamamoto, F., McNeill, P.D., and Hakomori, S. (1992). Human histo-blood group A2 transferase
coded by A2 allele, one of the A subtypes, is characterized by a single base deletion in the coding
sequence, which results in an additional domain at the carboxyl terminal. Biochem Biophys Res
Commun 187, 366-374.
Yamamoto, F., McNeill, P.D., Yamamoto, M., Hakomori, S., Harris, T., Judd, W.J., and Davenport,
R.D. (1993a). Molecular genetic analysis of the ABO blood group system: 1. Weak subgroups: A3
and B3 alleles. Vox Sang 64, 116-119.
Yamamoto, F., McNeill, P.D., Yamamoto, M., Hakomori, S., and Harris, T. (1993c). Molecular genetic
analysis of the ABO blood group system: 3. A(X) and B(A) alleles. Vox Sang 64, 171-174.
16. ABO Alleles (cis-AB & B(A) Alleles)
In addition to the weak subgroup alleles, we also characterized mutations in the alleles that specified
interesting phenomena of cis-AB and B(A). Cis-AB is a phenomenon where the expression of both A
and B antigens are specified by a single allele, as opposed to regular trans-AB, which is the
combination of A and B alleles from the father and mother. The cis-AB allele usually specifies a weak
expression of A and a weaker expression of B (phenotypically A2B3) (Yamaguchi et al., 1965, 1966).
B(A) was discovered when certain monoclonal anti-A antibodies were found to react with RBCs that
were previously typed as B (Treacy and Stroup, 1987). B(A) is similar to cis-AB in the sense that one
allele specifies the expression of both A and B antigens, however, B(A) specifies weak A antigen
expression in addition to strong B antigen expression. By PCR amplification of genomic DNA and
following DNA sequencing, we identified mutations in the cis-AB01 and B(A)01 alleles (Yamamoto et
al., 1993b; Yamamoto et al., 1993c). We found that the proteins encoded by these alleles are A-B
transferase chimeras. The cis-AB01 allele had a coding sequence identical to the A101 allele, except
for G268A (the last of the four amino acid substitutions that discriminate the A and B transferase is
alanine of B transferase). Conversely, the B(A)01 allele had a coding sequence identical to the B101
allele, except that the second of 4 amino acid substitutions is the amino acid of A transferase (G at
codon 235 in place of S).
Treacy, M., and Stroup, M. (1987). A scientific forum on blood grouping serum Anti-A (Murine
Monoclonal Blend) BioClone*. Raritan, NJ: Ortho Diagnostic Systems.
Yamaguchi, H., Okubo, Y., and Hazama, F. (1965). An A2B3 phenotype blood showing atypical mode
of inheritance. Proc Jpn Acad 41, 316-320.
Yamaguchi, H., Okubo, Y., and Hazama, F. (1966). Another Japanese A2B3 blood-group family with
the propositus having O-group father. Proc Jpn Acad 42, 517-520.
Yamamoto, F., McNeill, P.D., Kominato, Y., Yamamoto, M., Hakomori, S., Ishimoto, S., Nishida, S.,
Shima, M., and Fujimura, Y. (1993b). Molecular genetic analysis of the ABO blood group system: 2.
cis-AB alleles. Vox Sang 64, 120-123.
Yamamoto, F., McNeill, P.D., Yamamoto, M., Hakomori, S., and Harris, T. (1993c). Molecular genetic
analysis of the ABO blood group system: 3. A(X) and B(A) alleles. Vox Sang 64, 171-174.
17. ABO Alleles 2008
Since our description of the molecular genetic basis of the three major alleles and several minor
alleles, additional ABO alleles have been molecularly characterized and their sequence information
has
been
deposited
in
the
national
database (http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home), which was established
by Professor Olga Blumenfeld at Albert Einstein College of Medicine. Olsson, Chester, and
colleagues (Hansen et al., 1998; Olsson and Chester, 1995, 1996a, b, c, 1998, 2001; Olsson et al.,
1997; Olsson et al., 2001; Olsson et al., 1998; Olsson et al., 1995), Blancher and colleagues
(Kermarrec et al., 1999; Roubinet et al., 2002; Roubinet et al., 2001), Seltsam, Blasczyk, and
colleagues (Seltsam et al., 2002; Seltsam et al., 2003), Ogasawara and colleagues (Ogasawara et
al., 1996a; Ogasawara et al., 1998; Ogasawara et al., 2001; Ogasawara et al., 1996b), and Yip and
colleagues (Yip, 2000, 2002; Yip et al., 1996; Yip et al., 1995) played an important role in this
expansion. The total number of defined alleles currently stands at 178, and the full listing of alleles is
shown above. This table was prepared based on data culled from the dbRBC - Blood Group Antigen
Gene Mutation Database.
Blumenfeld,
O.
dbRBC
-
Blood
Group
Antigen
Gene
Mutation
Database (http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home)
Hansen, T., Namork, E., Olsson, M.L., Chester, M.A., and Heier, H.E. (1998). Different genotypes
causing indiscernible patterns of A expression on A(el) red blood cells as visualized by scanning
immunogold electron microscopy. Vox Sang 75, 47-51.
Kermarrec, N., Roubinet, F., Apoil, P.A., and Blancher, A. (1999). Comparison of allele O sequences
of the human and non-human primate ABO system. Immunogenetics 49, 517-526.
Ogasawara, K., Bannai, M., Saitou, N., Yabe, R., Nakata, K., Takenaka, M., Fujisawa, K., Uchikawa,
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(2001). Recombination and gene conversion-like events may contribute to ABO gene diversity
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18. ABO Alleles (BGAGMD)
The ABO alleles were aligned by their names. Only the differences (mutations/single nucleotide
polymorphisms (SNPs)) from the A101 allele are shown on the appropriate nucleotide locations (5' to
3' of the gene is shown from left to right). The exon sequences are shown in dark gray and the intron
sequences are shown in light gray. The locations of the four amino acid substitutions that discriminate
A and B transferases are indicated by red arrows, and the O-allele specific single nucleotide deletion
at nucleotide 261 is shown with a blue arrow. The small area of the table was enlarged and is shown
in the bottom panel.
This table was modified from the table prepared by Professor Blumenfeld (BGAGMD).
19. ABO Allele Mutations
Important mutations in human ABO genes are categorized. Different molecular mechanisms may be
responsible for seemingly identical phenotypes. For example, the B3 phenotype may be caused by a
missense mutation (A301 allele: D291N), a splicing mutation (B303), or the combination of a
missense mutation and a single nucleotide deletion, possibly due to recombination (A302: V277M and
1060delC). The kinds of mutations responsible for the ABO subgroups range from missense
mutations (A202, A203, A207, A301, A303, Ax01, Ax07, Ax12, Ax13, Bx02, Bx03, etc.), frame-shift
mutations due to nucleotide deletions (O01, O02, A206, Ael03, etc.), frame-shift mutations due to
nucleotide insertions (Ael01, Bw20, etc.), splicing mutations (Ael04, B303, etc.), an initiation codon
mutation (Aw13), and recombination (A302, Aw07, etc.). Nonsense mutations have yet to be found. A
localization mutation has recently been found in 2008.
20. Transfection Analysis
A functional analysis was designed to examine the effects of the mutations we identified on the activity
and specificity of human A and B transferases. We constructed the expression constructs of A and B
transferases and their derivatives in the pSG-5 eukaryotic expression vector. We transfected DNA
from those constructs into the human cancer cell line HeLa cells of the uterus. These cell line cells
are derived from a type O individual and express H antigens on the cell surface. Therefore, it was
expected that if the constructs encode functional A/B transferases, A/B antigens would be produced
and expressed on the cell surface. These newly synthesized antigens would then be immunologically
detected using anti-A and anti-B antibodies.
21. Transfection Results (A/B transferase)
We initially constructed A101 and B101 constructs that encoded A1 and B transferases. When DNA
from those constructs was transfected, A and B antigens appeared respectively on the cell surface
as anticipated.
Yamamoto, F., and Hakomori, S. (1990). Sugar-nucleotide donor specificity of histo-blood group A
and B transferases is based on amino acid substitutions. J Biol Chem 265, 19257-19262.
(http://www.jbc.org/cgi/reprint/265/31/19257)
22. Transfection Results (A2 and A3 Alleles)
When A201 allele-specific mutations (P156L and 1060delC) or the single nucleotide deletion
(1060delC) were introduced into the A101 construct, A antigen expression did not disappear but did
diminish. The A301 allele-specific amino acid substitution (D291N) also diminished given A antigen
expression (Yamamoto, 1995).
Yamamoto, F. (1995). Molecular genetics of the ABO histo-blood group system. Vox Sang 69, 1-7.
23. Transfection Results (O Alleles)
When O01 allele-specific single nucleotide deletion (261delG) or O03 allele-specific amino acid
substitutions (R176G and G268R) were introduced into the functional A101 construct, A antigen
expression disappeared.
Yamamoto, F. (1995). Molecular genetics of the ABO histo-blood group system. Vox Sang 69, 1-7.
24. Transfection Results (A-B Transferase Chimeras --AA)
Next, we examined the effects of 4 amino acid substitutions that discriminated A and B transferases.
For this purpose, we constructed 14 chimeras that possessed amino acid residues of either A or B
transferases at the same four locations; R/G, G/S, L/M, and G/A at codons 176, 235, 266, and 268
(Yamamoto and Hakomori, 1990). The results are summarized as follows. When the last two amino
acids of the 4 amino acid substitutions were L and G of A transferase, the constructs specified the
expression of A antigens only.
Yamamoto, F., and Hakomori, S. (1990). Sugar-nucleotide donor specificity of histo-blood group A
and B transferases is based on amino acid substitutions. J Biol Chem 265, 19257-19262.
(http://www.jbc.org/cgi/reprint/265/31/19257)
25. Transfection Results (A-B Transferase Chimeras --BB)
Similarly, when the last two amino acids of the 4 amino acid substitutions were M and A of B
transferase, the constructs expressed only B antigens.
Yamamoto, F., and Hakomori, S. (1990). Sugar-nucleotide donor specificity of histo-blood group A
and B transferases is based on amino acid substitutions. J Biol Chem 265, 19257-19262.
(http://www.jbc.org/cgi/reprint/265/31/19257)
26. Transfection Results (A-B Transferase Chimeras --AB)
When they were chimeric (L of A transferase and A of B transferase), the results depended on the
amino acid residue at the second position. When it was G of A transferase, the constructs expressed
only A antigens. However, when the residue was S of B transferase, the constructs expressed small
amounts of B antigens in addition to A antigens.
Yamamoto, F., and Hakomori, S. (1990). Sugar-nucleotide donor specificity of histo-blood group A
and B transferases is based on amino acid substitutions. J Biol Chem 265, 19257-19262.
(http://www.jbc.org/cgi/reprint/265/31/19257)
27. Transfection Results (A-B Transferase Chimeras --BA)
Finally, when the last 2 amino acids were M of B transferase and G of A transferase, the constructs
strongly expressed both A and B antigens. This became the first demonstration where we successfully
modified the specificities of glycosyltransferases by genetic engineering and created novel enzymes
with both strong A and B transferase activities.
Yamamoto, F., and Hakomori, S. (1990). Sugar-nucleotide donor specificity of histo-blood group A
and B transferases is based on amino acid substitutions. J Biol Chem 265, 19257-19262.
(http://www.jbc.org/cgi/reprint/265/31/19257)
28. Transfection Results (A transferase Codon 268)
We further expanded the molecular enzymology study by constructing the in vitro mutagenized A and
B transferase constructs that possessed any one of the 20 amino acids at codon 268 (Yamamoto and
McNeill, 1996). A transferase possessed the smallest amino acid residue G at codon 268 and
expressed only A antigens. When it was replaced with A, the second smallest amino acid residue,
the construct expressed small amounts of B antigens in addition to A antigens. When it was replaced
with the slightly larger S or cysteine (C), the constructs also expressed smaller amounts of A antigens
and larger amounts of B antigens. The N and threonine (T) constructs only expressed small amounts
of B antigens. The remaining constructs did not express either A or B antigens with the exception of
the histidine (H) construct, which expressed somewhat moderate amounts of A antigens.
Yamamoto, F., and McNeill, P.D. (1996). Amino acid residue at codon 268 determines both activity
and nucleotide-sugar donor substrate specificity of human histo-blood group A and B transferases: In
vitro
mutagenesis
study.
J
(http://www.jbc.org/cgi/content/full/271/18/10515)
Biol
Chem
271,
10515-10520.
29. Nucleotide-Sugar Specificity (A transferase Codons 266-268)
The area of the A transferase surrounding codons 266 and 268 is schematically shown. With the
original glycine (G) residue at codon 268, only a GalNAc fits well to the cavity space. When it is
replaced by the alanine (A) residue, the space becomes a little smaller. As a result, not only a GalNAc
but also a galactose is able to fit in this space. The situation is the same for the replacement of the
glycine residue with serine (S) or cysteine (C), but when it is replaced by a larger residue, neither a
GalNAc nor a galactose fits in the space any longer.
30. Transfection Results (B transferase Codon 268)
The original B transferase construct possessed A and expressed B antigens. In the in vitro enzymatic
assays, weak activity of A transferase was also detected as previously reported (Greenwell et al.,
1986). When it was replaced with smaller G, the construct expressed both A and B antigens. When it
was replaced with slightly larger S or C, the constructs still exhibited strong B antigen expression.
When it was replaced with larger N or T, the constructs exhibited decreased B antigen expression.
When the N and D constructs were compared with the glutamine (Q) and glutamic acid (E) constructs,
the expression of B antigen was stronger with the N and D constructs than the Q and E constructs.
Similarly, when the N construct was compared with the D construct or when the Q construct was
compared with the E construct, the expression of the B antigen was stronger with the N construct
than with the D construct and also stronger with the Q construct than with the E construct. Taken
together, it was concluded that the size and charge of the amino acid residue at codon 268 was crucial
in determining the activity and specificity of the glycosyltransferases. Because the results differed
between the constructs with the A transferase backbone and those with the B transferase backbone,
it was also shown that not only codon 268, but also amino acid substitutions at other 3 locations, are
important, with codon 266 most likely being of primary importance.
Yamamoto, F., and McNeill, P.D. (1996). Amino acid residue at codon 268 determines both activity
and nucleotide-sugar donor substrate specificity of human histo-blood group A and B transferases: In
vitro mutagenesis
study.
J
Biol
(http://www.jbc.org/cgi/content/full/271/18/10515)
Chem
271,
10515-10520.
31. Nucleotide-Sugar Specificity (B Transferase Codons 266-268)
The area of the B transferase surrounding codons 266 and 268 is schematically shown. With the
original alanine (A) at codon 268, a galactose fits well to the cavity space. When the alanine is
replaced by the glycine (G) residue, the space becomes bigger and either a GalNAc or a galactose
fits within the space. When the alanine is replaced by bigger amino acids, the efficiency becomes
lower depending on their sizes. The charge of the amino acid at codon 268 is also important.
32. Three-Dimensional Structure of A Transferase
Our prediction that the amino acid residues at codons 266 and 268 are directly involved in the
recognition and binding of the sugar portion of donor nucleotide-sugar substrate in the transferase
reaction, whereas the amino acid residue at codon 176 is far from the site and the amino acid residue
at codon 235 is in-between, was later proven correct by the plotting of the 3-D structure of crystallized
human A transferase by others (Patenaude et al., 2002).
Patenaude, S.I., Seto, N.O., Borisova, S.N., Szpacenko, A., Marcus, S.L., Palcic, M.M., and Evans,
S.V. (2002). The structural basis for specificity in human ABO(H) blood group biosynthesis. Nature
Struct Biol 9, 685-690.
33.Homologous Sequences in Other Species
Once we cloned cDNAs encoding the human A transferase, we used one of these cDNAs to probe
homologous sequences in the genomes of other species of organisms. We observed weak signals in
chicken genomic DNA and strong signals in all mammalian species tested (Kominato et al., 1992).
Surprisingly, we found strong genomic DNA signals in dog, cat, and rabbit samples – signals as strong
as those in humans.
Kominato, Y., McNeill, P.D., Yamamoto, M., Russell, M., Hakomori, S., and Yamamoto, F. (1992).
Animal histo-blood group ABO genes. Biochem Biophys Res Commun 189, 154-164.
34. Primate ABO Gene Sequence Comparison
We also determined partial nucleotide sequences of ABO genes from several species of primates
(chimpanzee A, gorilla B, orangutan A, macaque A, and baboon A and B). When we compared those
primate sequences with human sequences, we found that the last two of four amino acid substitutions
that discriminated human A and B transferases were conserved depending on the A/B phenotype;
they are L and G in A alleles and M and A in B alleles.
Kominato, Y., McNeill, P.D., Yamamoto, M., Russell, M., Hakomori, S., and Yamamoto, F. (1992).
Animal histo-blood group ABO genes. Biochem Biophys Res Commun 189, 154-164.
35. Evolution of ABO Genes in Primates
We constructed a phylogenetic tree of the primate ABO genes and obtained results that suggested
that A to B changes might have occurred in at least three separate occasions during the evolution of
ABO genes (Saitou and Yamamoto, 1997).
Saitou, N., and Yamamoto, F. (1997). Evolution of primate ABO blood group genes and their
homologous genes. Mol Biol Evol 14, 399-411. (http://mbe.oxfordjournals.org/cgi/reprint/14/4/399)
36. Evolution of ABO Genes (2008)
The human ABO genes and their orthologous genes from other species were extracted from the
Ensembl database, and a phylogenetic tree was constructed. There are 46 genes from 23 species.
Ensembl Human Database (http://www.ensembl.org/Homo_sapiens/index.html)
37. Alpha 1-3 Gal(NAc) Transferase Family
The family of α1-3 Gal(NAc) transferases include A and B transferases, α1-3 Gal transferase,
isogloboside 3 synthase, and Forssman glycolipid synthase. A transferase and Forssman glycolipid
synthase transfer a GalNAc, whereas B transferase, α1-3 Gal transferase, and isogloboside 3
synthase transfer a galactose.
38. Evolutionary Tree of ABO and Related Genes (2001)
In addition to the analysis of primate ABO genes, we also cloned cDNAs from porcine and murine
ABO genes (Yamamoto and Yamamoto, 2001; Yamamoto et al., 2001). We demonstrated that the
murine gene is well-conserved within the species and encodes an enzyme with both A and B
transferase activity in the in vitro assay (although A antigens are primarily synthesized in vivo). Pigs
(porcine genes) exhibit AO polymorphism. Our study showed that the most, if not all of the structural
gene is missing in type O pigs, and therefore, inadvertent expression of an A antigen in
xenotransplanted tissues/organs from O pigs will be unlikely. Based on our studies, we constructed
an evolutionary tree of ABO and related genes. Those related genes included α1-3 Gal transferase
genes that synthesize α1-3 Gal epitope from mouse, pig, and cow samples, and a Forssman synthase
gene that had been cloned from dog by that time. The murine and porcine ABO genes clustered with
the human ABO genes, rather than with the α1-3 Gal transferase genes and the Forssman synthase
gene.
Yamamoto, F., and Yamamoto, M. (2001). Molecular genetic basis of porcine histo-blood group AO
system. Blood 97, 3308-3310. (http://bloodjournal.hematologylibrary.org/cgi/reprint/97/10/3308)
Yamamoto, M., Lin, X.H., Kominato, Y., Hata, Y., Noda, R., Saitou, N., and Yamamoto, F. (2001).
Murine equivalent of the human histo-blood group ABO gene is a cis-AB gene and encodes a
glycosyltransferase with both A and B transferase activity. J Biol Chem 276, 13701-13708.
(http://www.jbc.org/cgi/content/full/276/17/13701)
39. Partial Amino Acid Sequence Comparison (2001)
The deduced amino acid sequences surrounding codons 266 and 268 of the human A and B
transferases and corresponding sequences of evolutionarily-related genes are shown. The asterisks
indicate the positions of codons 266 and 268. Human A transferase has leucine (L) and glycine (G)
at those positions, whereas human B transferase has methionine (M) and alanine (A). The human
O03 allele has leucine and arginine (R), and because of the arginine, the protein has no enzymatic
activity. The cis-AB01 allele is an A-B transferase chimera and has leucine of A transferase and
alanine of B transferase. The mouse AB gene has alanine at codon 268 as in human B transferase,
but contains glycine, which is much smaller than methionine of human B transferase. This allows the
transfer of both a GalNAc and a galactose in the in vitro transfection experiments using human HeLa
cells as the host. However, A antigens are primarily synthesized in vivo. Pig A gene has alanine and
glycine at codons 266 and 268. Because the glycine at codon 268 is the same as in human A
transferase and the alanine at codon 266 is smaller than the leucine in human A transferase, the
enzyme transfers only a GalNAc. Dog Forssman glycolipid synthase has glycine and alanine, the
same two amino acids as the mouse cis-AB gene. However, possibly because valine at codon 269 is
smaller than phenylalanine (F), the protein transfers only a GalNAc. Both murine and bovine α1-3 Gal
transferases have histidine (H) and alanine. Because alanine is the same as alanine in the human B
transferase and histidine is bigger than methionine, they transfer only a galactose.
Yamamoto, F., and Yamamoto, M. (2001). Molecular genetic basis of porcine histo-blood group AO
system. Blood 97, 3308-3310. (http://bloodjournal.hematologylibrary.org/cgi/reprint/97/10/3308)
Yamamoto, M., Lin, X.H., Kominato, Y., Hata, Y., Noda, R., Saitou, N., and Yamamoto, F. (2001).
Murine equivalent of the human histo-blood group ABO gene is a cis-AB gene and encodes a
glycosyltransferase with both A and B transferase activity. J Biol Chem 276, 13701-13708.
(http://www.jbc.org/cgi/content/full/276/17/13701)
40. Evolutionary Tree of ABO and Related Genes (2008)
Thanks to the Human Genome Project and the genome sequencing projects of other species of
organisms, the number of other sequenced ABO and related genes has increased to 144 genes from
31 species. A phylogenetic tree of those genes was constructed and is shown. In addition to the ABO
genes (A/B transferases), GGTA1 genes (α1-3 Gal transferases), A3GALT2 genes (isogloboside 3
synthases), and GBGT1 genes (Forssman glycolipid synthases) previously identified, another group
of genes formed a cluster and were named as GLT6D1 (glycosyltransferase 6 domain containing 1)
genes. It remains to be determined whether any genes in this group may encode active enzymes that
transfer galactose, GalNAc, or both by α1-3 glycosidic linkage.
Ensembl Human Database (http://www.ensembl.org/Homo_sapiens/index.html)
41. Partial Amino Acid Sequence Comparison (2008)
The right portion shows the deduced amino acid sequences around the amino acid residues
corresponding to codons 266 and 268 of the human A/B transferases. The sequences are wellconserved, especially those around the 2 amino acid residues. The locations of codons corresponding
to codons 266 and 268 of human A and B transferases are indicated by asterisks.
Ensembl Human Database (http://www.ensembl.org/Homo_sapiens/index.html)
42. Partial Amino Acid Sequence Comparison (GGTA1 Genes)
For the GGTA1 genes, the sequence of GDFYYHAA(I/L/V)FGG is highly conserved.
Ensembl Human Database (http://www.ensembl.org/Homo_sapiens/index.html)
43. Partial Amino Acid Sequence Comparison (A3GALT2 Genes)
For the A3GALT2 genes, the sequence of GDFYYHAA(I/L/V)FGG is also highly conserved. Rabbit
(GDFYYHATL--G) and cat (GDFYYHTAVFGG) A3GALT2 genes were the only exceptions among the
species whose gene sequences were determined. Two findings suggest that the A3GALT2 genes in
these two species are non-functional. The first is the deletion of two amino acid residues (--) inside
the conserved sequence in the rabbit sample. The second is the longer deletions downstream of
these conserved sequences in both rabbit and cat genes.
Ensembl Human Database (http://www.ensembl.org/Homo_sapiens/index.html)
44. Partial Amino Acid Sequence Comparison (GBGT1 Genes)
For the GBGT1 genes, the sequence of the same location is also highly conserved. However, the
conserved sequence is GDFYYGGA(L/V)FGG, and the HA sequence in the GGTA1 and A3GALT2
transferases is replaced with GG in the transferases. The exceptions were found in macaque and
cow (GDFYYGRAVFGG) genes. Ten amino acids are deleted just downstream of this sequence in
cow, which explains why cow is Forssman-negative. The inactivity is also explained by the amino acid
substitution of alanine to lysine (A268K) in the macaque gene. This finding is based on the results of
the A268K in vitro mutagenized construct of human B transferases.
45. Partial Amino Acid Sequence Comparison (ABO Genes)
In contrast to the 3 glycosyltransferase genes of GGTA1, A3GALT2, and GBGT1, there are more
variations among the genes in the ABO gene family. In addition to the combinations of L and G for
codons 266 and 268 in human A transferase and M and A in human B transferase, there are
additional amino acid combinations. These include A and G in dogs and cats and T and A in greater
galagos and hedgehogs. Whether proteins with other combinations exhibit functional transferase
activity remains to be determined.
46. Partial Amino Acid Sequence Comparison (GLT6D1 Genes)
There is wide variation in codons 266 and 268 in the GLT6D1 genes. We do not know whether any
of the GLT6D1 genes encode functional glycosyltransferases or not.
Ensembl Human Database (http://www.ensembl.org/Homo_sapiens/index.html)
47. Glycosyltransferase Gene Family
The biosynthesis of A and B antigens are catalyzed by A and B transferases, respectively. However,
the common acceptor substrate of the H antigen is also produced by the reaction catalyzed by another
glycosyltransferase, α1-2 fucosyltransferase. Oligosaccharide structures are generally synthesized
through a series of reactions catalyzed by several glycosyltransferases, rather than a single reaction.
It is, therefore, necessary to study the expression of several, if not many, glycosyltransferases in order
to understand the expression of certain oligosaccharide antigens. For this purpose, we developed an
experimental system to study the expression of 68 human glycosyltransferase genes (Yamamoto et
al, 2003). In humans α1-3 Gal(NAc) transferase genes other than ABO genes are non-functional or
have become pseudogenes during evolution. Therefore, only one gene, which is indicated by an
arrow, represents this family. As you see, there are many genes encoding glycosyltransferases. They
were categorized depending on which sugar is transferred. It should be noted that this table does not
cover all the glycosyltransferases, though the majority of the enzymes are included.
Yamamoto, M., Yamamoto, F., Luong, T.T., Williams, T., Kominato, Y., Yamamoto, F. (2003).
Expression profiling of 68 glycosyltransferase genes in 27 different human tissues by the systematic
multiplex reverse transcription-polymerase chain reaction method revealed clustering of sexually
related tissues in hierarchical clustering algorithm analysis. Electrophoresis 24, 2295-2307.
48. Glycosyltransferase Gene Expression in Human Tissues
Using this system, we examined the expression of those genes in 27 different tissues by the technique
which we named Systematic Multiplex RT-PCR (SM RT-PCR). The panoramic view of a total of 1836
(68x27) expression data demonstrates that some glycosyltransferase genes are differentially
expressed, whereas some others are ubiquitously expressed. Although the expression profiling of
glycosyltransferase genes alone may not directly explain the repertoires of oligosaccharides
synthesized, it is an important step toward a better understanding of the gene expression network
involved in oligosaccharide synthesis/degradation.
Yamamoto, M., Yamamoto, F., Luong, T.T., Williams, T., Kominato, Y., Yamamoto, F. (2003).
Expression profiling of 68 glycosyltransferase genes in 27 different human tissues by the systematic
multiplex reverse transcription-polymerase chain reaction method revealed clustering of sexually
related tissues in hierarchical clustering algorithm analysis. Electrophoresis 24, 2295-2307.
49. Glycosyltransferase Gene Expression Analyzed by Hierarchical Clustering Algorithm
Our modestly high-throughput gene expression study and data analysis using a hierarchical clustering
algorithm have allowed us to investigate the correlation between tissues and glycosyltransferase gene
expression. Similar patterns of glycosyltransferase gene expression were observed in functionallyand anatomically-related tissues. All but one of the sexually-related tissues formed a cluster in a tissue
dendrogram, suggesting the involvement of sex hormones in the transcriptional control of many
glycosyltransferase genes. Once established, the SM RT-PCR is cost-effective and time-efficient, and
requires small amounts of RNA as a template. It is especially useful for the simultaneous analyses of
multiple samples. Because of its simple design, the SM RT-PCR may offer an easy alternative in
studying the expression of many other families of genes, as well as groups of related/unrelated genes
in various biological phenomena.
Yamamoto, M., Yamamoto, F., Luong, T.T., Williams, T., Kominato, Y., Yamamoto, F. (2003).
Expression profiling of 68 glycosyltransferase genes in 27 different human tissues by the systematic
multiplex reverse transcription-polymerase chain reaction method revealed clustering of sexually
related tissues in hierarchical clustering algorithm analysis. Electrophoresis 24, 2295-2307.
50. Summary
Due to time restriction, the studies regarding genomic DNA cloning and the elucidation of gene
organization and gene expression controlling mechanisms have not been mentioned in this talk.
Appendix 01. Discovery of the ABO blood group system
This slide shows the results of an experiment that mixed the cellular and liquid components of blood.
Landsteiner separated the cellular and liquid components of blood from both his colleagues and
himself, and mixed them in different combinations. He then observed the agglutination of red blood
cells (RBC) in certain combinations, as indicated by the plus (+) symbols in the table. He also
observed the absence of agglutination in the other combinations, as indicated by the minus (-)
symbols. When cellular and liquid components from the same individuals were combined, no RBC
agglutination was observed. If RBC agglutination occurs in the human body, it was expected that
capillaries would be clogged and adverse effects would be elicited. Therefore, Landsteiner’s
experiment showed for the first time that blood transfusion has to be performed in a combination
where no RBCs are agglutinated. This discovery subsequently led to the development of safe medical
practices regarding blood transfusion. Additionally, the results also showed that individuals can be
grouped based on agglutination patterns. In the table shown here, Dr. Pleen. and Mr. Zar. belong to
one group, Dr. Sturl. and Dr. Erdh. belong to another, and Dr. St. and Mr. Landsteiner belong to the
third group. In the following year, the fourth group was found by Landsteiner’s disciples, and these 4
groups became the ABO blood group system.
Appendix 02. Four major groups
Four major groups of A, B, AB, and O are defined by the presence or absence of 2 antigens (A and
B) on RBCs, and of the antibodies against these antigens in sera. Blood group A individuals have A
antigens on RBCs and anti-B antibodies in serum. Similarly, blood group B individuals have B antigens
on RBCs and anti-A antibodies in serum. Blood group AB individuals have both A and B antigens on
RBCs and neither anti-A nor anti-B antibodies in serum. On the contrary, blood group O individuals
have neither A antigens nor B antigens, but possess both anti-A and anti-B antibodies in serum. The
observed rule that individuals have the antibodies against A or B antigens if they do not express A or
B antigens on RBCs, respectively, has been named the Landsteiner’s Law. The presence or absence
of A or B antigens can be detected by the RBC agglutination reaction, using the anti-A or anti-B
reagents (the forward test). The presence or absence of anti-A or anti-B antibodies in serum can be
detected by the RBC agglutination reaction, using the reference A1 and B RBCs (the reverse test).
These tests are routinely used to determine ABO blood type at any hospital.
Appendix 03. Genetic basis of ABO blood grouping
In 1910, von Dungern and Hirszfeld hypothesized that ABO blood groups are characteristics that are
inheritable from generation to generation. Later, in 1924, Bernstein proposed the one genetic locus–
three allelic model, which explained the mode of inheritance of the ABO blood groups. He postulated
that there were three alleles (A, B, and O) at the single genetic locus of ABO. He assumed that A and
B alleles are co-dominant against the recessive O allele. Based on the combination of two alleles,
there are six genotypes of AA, AO, BB, BO, AB, and OO, which result in 4 phenotypes of A, B, AB,
and O. Several examples of inheritance from parents of certain genotypes are also shown. For
example, from the parents of AA and AO, only the children with AA or AO genotypes are possible.
Similarly, from the parents of AB and O (OO genotype) phenotypes, only the children with A (AO) or
B (BO) phenotypes are possible, and no children with AB or O phenotypes are expected (See the cisAB cases for exception).
Appendix 04. ABH(O) substances
A and B antigens were initially identified on RBCs. However, antigens with similar specificities were
later identified in secretions such as saliva, seminal fluid, and ovarian cyst fluid. In addition to RBCs,
they were also found on the other types of cells (endothelial cells and epithelial cells). It also became
clear that the expression of these antigens is not restricted to humans, but also exists in other species
of living organisms.
Appendix 05. Immuno-determinant structures of ABH(O) antigens
The chemical structures of the A and B antigens are shown. Two major groups: Watkins and Morgan
in London, and Kabat and colleagues in New York, contributed to the chemical characterization.
These antigens are not protein antigens but oligosaccharide antigens. Certain sugar residues are
bound by specific linkages to form special structures in these antigens. More specifically, the immunodominant structure of the A antigen is specified by the presence of a fucose residue and a GalNAc
residue attached to a galactose by alpha 1,2- and alpha 1,3 glycosidic linkages. The galactose residue
is also linked to the internal core structure. In the B antigens, the GalNAc residue in the A antigen is
replaced by a galactose residue.
A structurally similar antigen was found to be abundantly present
in O individuals, and was named the H antigen. The immuno-dominant structure of the H antigen
lacks both the GalNAc in the A antigen and the galactose in the B antigen linked to galactose by alpha
1,3 linkage.
Appendix 06. The immuno-dominant sugars of the A and B antigens are GalNAc (N-acetyl-Dgalactosamine) and galactose, respectively.
The chemical structures of these sugars are shown. The only difference between these two sugars is
the N-acetyl group (-NHCOCH3) and the hydroxyl group (-OH), which are attached at the 2nd position
of the carbon ring in GalNAc and galactose, respectively.
Appendix 07. The Biosynthetic pathways of the A and B antigens
In 1959, Watkins and Morgan, and separately Ceppellini, proposed a hypothesis regarding the
biosynthetic pathways of A and B antigens.
In this hypothesis, they postulated that the two
glycosyltransferases (A and B transferases) encoded by the functional alleles (A and B alleles)
catalyze the last step of biosynthesis. It was assumed that A transferase transfers a GalNAc residue
to the acceptor H structure by alpha 1,3 glycosidic linkage, whereas B transferase transfers a
galactose residue in place of a GalNAc.
Appendix 08. Linkage analysis of the ABO genes
Renwick and Lawler observed the association of the ABO blood groups with the Nail-Patella
syndrome in 1955. This disease is characterized by several typical abnormalities of the arms and legs
as well as kidney disease and glaucoma, and is inherited in an autosomal dominant manner. The
close linkage of the ABO blood group and the disease indicated that the ABO gene locus and the
genetic loci to cause the syndrome are located in the same chromosomal region. Ferguson-Smith
and colleagues later mapped the ABO gene locus to 9q34, using somatic cell hybrids.
Appendix 09. ABO blood groups and A and B transferase activity
A and B transferase activities were measured using sera and tissues from individuals with different
ABO blood groups. It was found that A transferase activity was present in biological specimens from
individuals with either an A or AB blood group, whereas B transferase activity was observed in
specimens from individuals with either the B or AB blood group. Neither A nor B transferase activity
was found in specimens from the O blood group individuals.
Appendix 10. History of the purification attempt of A transferase
The purification of A/B transferase has been attempted since 1970.
Sources rich in A/B transferase
activity such as human milk and plasma were used. Although the attempts had some success in
identifying the fractions that were enriched with enzyme activity, they were not pure. In 1990, Henrik
Clausen, a colleague of mine at the now defunct Biomembrane Institute, obtained two fractions of
pure proteins from human lungs that seemed to be the soluble form of A transferase. The reversephase chromatography used at the last step of the purification procedures denatured the isolated
proteins, and no enzymatic activity was observed. The partial amino acid sequences of one of the
proteins revealed that the protein was the cystic fibrosis antigen protein.
Apparently, one or several
of the patients whose lungs were used for the purification seemed to have suffered from the disease.
The partial amino acid sequences for the other proteins were not found in the DNA and protein
sequence databases. If the activity was not lost during the last step, the specific activity was calculated
to be high (5.7 units/mg).
Appendix 11. A and B subgroups
This table lists most of the A and B subgroups. Depending on the strength and pattern of RBC
agglutination with the typing reagents, A and B groups can be further divided into subgroups. Other
criteria such as the presence/absence of unexpected anti-A or anti-B antibodies, as well as the
presence/absence of ABH substances in saliva, are also used. A1 occupies the majority of A
individuals. Other A subgroups include A2, A3, Ax, and Ael. Similarly, B (B1) occupies the majority of
B individuals, and other B subgroups include B3, Bx, and Bel.
Appendix 12. A1 and A2 subgroups
Two subgroups of A1 and A2 were initially discriminated by von Dungern in 1911. Dolichos biflorus
plant lectin or the anti-A1 antibody that was prepared by pre-treating serum from blood group B
individuals with A2 RBCs agglutinates A1 RBCs, whereas no agglutination was observed with A2
RBCs. Later studies have found that the number of A antigens is much higher with A1 RBCs than A2
RBCs. It was also shown that A1 RBCs possess A antigens on the branched and unbranched core
structures, whereas A2 RBCs have antigens predominantly on unbranched structures. Additionally,
more recent studies have shown the presence of A antigens on the type 4 core structure only on A1
RBCs.
Appendix 13. A3, Ax, and B3 weak subgroups
Some of the most important characteristics of the three weak subgroups (A3, Ax, and B3) are shown.
A3 and B3 phenotypes are most well-characterized by mixed-field agglutination patterns under the
microscope, where some cells exhibit agglutination and others do not. The Ax RBCs are not
agglutinated by anti-A reagents, but are weakly agglutinated with anti-A,B reagents. There seems to
be heterogeneity among the cases in the same subgroups.
Appendix 14. Discovery of cis-AB
In 1964, Seyfried reported a family case where the inheritance of ABO blood groups did not follow the
one genetic locus-three allelic model proposed by Bernstein. The ABO blood types of the father and
mother were O and AB, respectively, but those of their two children were AB. The blood type of the
mother’s mother was O. The next year Yamaguchi and colleagues reported an A2B3 phenotype. The
family study suggested that the A2B3 phenotype was inherited in the cis manner, and they named
this allele the cis-AB allele. As opposed to the regular trans-type of AB where the A allele is derived
from one parent and the B allele is derived from the other parent, the cis-AB behaves as one unit and
transcends from one parent to the next generation. Yoshida and colleagues characterized A and B
transferase activity in the sera of the cis-AB individuals, and proposed that there are two discrete
molecular mechanisms of the occurrence of the cis-AB allele: either crossing over, resulting in 2
alleles on a chromosome, or structural mutations leading to the production of an enzyme with
bifunctional activity. Our molecular study has identified structural mutations in several cis-AB alleles.
Appendix 15. Two examples of cis-AB inheritance
The cis-AB inheritance of the ABO blood group is shown with two family cases. In Family 1, the father
was phenotypically A2B3. The mother was O (OO) and the daughter, one of the children, was
phenotypically A2B3. This can be explained by assuming that the father’s ABO genotype was cisAB/O, and that the daughter inherited the cis-AB allele from her father and the O allele from her
mother, resulting in the genotype of cis-AB/O, the same genotype as her father.
In Family 2, the
ABO phenotypes of the father, mother, and son were A1, A1B3, and A2B3, respectively. This
inheritance can be explained by assuming that the mother’s genotype was cis-AB/A1, and the son
inherited the O allele from his father and the cis-AB allele from his mother to be cis-AB/O.
Appendix 16. Discovery of B(A) phenotype
The B(A) phenotype was discovered after murine monoclonal anti-A and anti-B antibodies were
introduced as anti-A and anti-B reagents. Treacy and Stroup reported in 1987 that some of the RBCs
previously typed as B reacted weakly with certain lots of murine monoclonal anti-A antibodies. They
named the phenotype B(A).
Appendix 17. Mapping of the ABO gene using the radiation hybrid panel
In 1996, we mapped the ABO gene using the radiation hybrid panel. The ABO gene was mapped
close to the end of the q-arm of chromosome 9. Actually, the gene was located closest to the end
compared with the all the marker sequences mapped in the region at that time. The current version
of the DNA database lists 120 genes between the ABO locus and the telomere.
Appendix 18. ABH and related antigens
The chemical structures of the ABH antigens and two additional antigens (alpha 1,3 galactosyl epitope
and Forssman antigen) are shown. As opposed to the ABH antigens, which possess a fucose linked
to the galactose by alpha 1,2 glycosidic linkage, the alpha 1,3 galactosyl epitope and Forssman
antigen do not possess the fucose. The A antigen and Forssman antigen possess a GalNAc linked to
the galactose by alpha 1,3 linkage, whereas the B antigen and alpha 1,3 galactosyl epitope possess
a galactose linked to the galactose by alpha 1,3 linkage.
Appendix 19. The genomic structure of the human ABO gene
We cloned genomic DNA fragments containing the ABO gene in 1995. The gene spans more than 18
kilobases, and the coding sequence scatters over 7 exons, with the longest coding sequence in the
last exon. We determined the exon-intron boundaries, and found that they all follow the GT-AG
splicing rule. The 3’ untranslated region is rich with repeat sequences, and may be involved in the
stability of the messages. We also delineated the promoter region of the ABO gene. Kominato played
the major role in the promoter characterization. To do so, we constructed a luciferase reporter
construct with the potential promoter region of the ABO gene that precedes the transcription initiation
codon, as well as different sizes of nested deletion. We determined the promoter activity of the
constructs and located the region with the promoter activity. The promoter is rich with CG, and there
is an enhancer element further upstream.
Appendix 20. Comparison of amino acid sequences of the ABO and related genes
The deduced amino acid sequences are compared among the human A (A101), B (B101), O (O03),
and cis-AB genes, mouse cis-AB gene, pig A gene, mouse and bovine alpha 1,3 galactosyltransferase
genes, and dog Forssman synthase gene. They exhibited strong homology, suggesting that these
genes are derived from a common ancestral gene.
Appendix 21. Comparison of gene organization between human and mouse ABO genes
The exon-intron boundaries of the human and mouse ABO genes were aligned and are shown.
Except for the N-terminal several amino acid residues that were scattered in 2 or 3 exons in the mouse
gene, both the human and mouse gene sequences corresponded quite well.
Appendix 22. Polymorphism in the ABO gene that was observed among different species and
subspecies of mice
We determined the partial nucleotide sequences of the mouse ABO genes from a variety of
subspecies of mice (Mus musculus). Saitou played the major role in the characterization. We also
determined the gene in another species of mouse (M. specifegus). We observed single nucleotide
polymorphism at several positions.
Appendix 23. The specificity of the murine enzyme
We constructed a cDNA expression construct of the mouse ABO gene and determined specificity. We
also constructed the Human-mouse chimeric construct by replacing the last coding exon of the human
construct with the mouse counterpart. Four constructs were used as controls: no DNA, pA(arg),
pAAAA, and pBBBB. The pA(arg) construct encodes A transferase, except that codon 268 was
replaced from glycine to arginine. Due to the amino acid substitution, this construct cannot exhibit A
transferase activity, and therefore was used as a negative control. pAAAA and pBBBB constructs
encode functional A and B transferases, respectively. Therefore, they were used for positive controls.
These constructs were transfected to the HeLa cell, the human uterine cancer cells, and the
appearance of A and B antigens were monitored by the immunological method using anti-A and antiB antibodies. Both the pHuman-mouse and pMouse constructs induced the expression of A and B
antigens, suggesting that the mouse gene is a cis-AB gene and encodes an enzyme with both A and
B transferase activity.
Appendix 24. Porcine ABO gene
Using the degenerate oligonucleotide primers, we amplified the partial sequence from the pig ABO
gene. We later used the sequence information to clone the pig ABO gene cDNA. The pig gene
encoded a functional enzyme with A transferase activity. Porcine A and O phenotypes were already
known. Therefore, we determined the A/O phenotype of the salivary gland tissues of pigs, and isolated
genomic DNA from the pig tissues exhibiting A and O phenotypes. DNA was cleaved with restriction
enzymes, electrophoresed, and Southern transferred onto a nylon membrane. After fixation, the filter
was hybridized with the radio-labeled pig A transferase gene fragment. The results of hybridization
demonstrated that genomic DNA derived from the pig with the O phenotype lacked the homologous
sequence. This is in contrast to the human O alleles, which either possess a single nucleotide deletion
that causes a frameshift or amino acid substitutions that nullify the enzymatic activity. Apparently, the
pig O allele seems to be lacking most, if not all, of the structural gene encoding the glycosyltransferase.
Appendix 25. A variety of methods for the ABO genotyping
Before our elucidation of the molecular genetic basis of the human ABO blood group system in 1990,
it was impossible to discriminate RBCs from individuals with an AA genotype from RBCs taken from
individuals with an AO genotype. The determination of the nucleotide sequences of the ABO alleles
and the identification of a single nucleotide polymorphism among those alleles allowed us to genotype
the ABO genetic locus. We used two different methods in the original study. We used the restriction
fragment length polymorphism (RFLP) followed by Southern hybridization with the human A
transferase probe to discriminate both the alleles with the O allele-specific single nucleotide deletion
(to be cleaved with KpnI) and the alleles without the deletion (to be cleaved with BstEII). We used
PCR to amplify the DNA fragments that contained the differences in the nucleotide sequence between
the A/O and B alleles. The amplified DNA fragments were later subjected to allele-specific restriction
enzyme digestion (BssHII and AluI for the A/O alleles and NarI and HpaII for the B allele). We later
performed successful allele-specific PCR using fluorescence-labeled primers. However, we decided
not to publish the results because Hood and colleagues reported the use of LCR (ligase chain
reaction) to discriminate ABO alleles using fluorescence detection. Later, a variety of methods were
also applied to ABO genotyping to detect the single nucleotide polymorphism.
Appendix 26. ABO Polymorphism & Infectious Disease Susceptibility
ABO Polymorphism & Disease Susceptibility
Infectious Diseases (1)
Genetic polymorphisms may have played a critical role in defending humans against pathogen
invasion. A classic example on how variability is of such importance is the major histo-compatibility
complex (MHC) genes, which encode for the proteins that present peptide-antigens to T-cells.
Another example is the ABO polymorphism which specifies the end product of specific
oligosaccharide antigens. Because ABH antigens are expressed on the surface of various types of
cells and are also secreted, differential interactions with infectious pathogens have been suggested,
in the past, by association studies and by binding experiments in recent years. Pathogens may have
carbohydrate-binding proteins, glycosyltransferases, and/or glycosidases that recognize and bind to
ABH antigens, and the host’s ABO phenotype may potentially affect the pathogen adhesion and
invasion.
It may also change the constituency of resident bacterial flora in the intestine, and modify
the immune response of the host. Additionally, blood group-like antigens are present on some bacteria,
and they may interact with host proteins with carbohydrate-binding capacity, such as selectins,
galectins, and siglecs.
They may also interact with naturally occurring antibodies.
A.E. Mourant summarized previous results of association studies of diseases, including infectious
diseases, with ABO blood group polymorphism in the Oxford Monographs on Medical Genetics (Blood
Groups and Diseases: A study of associations of diseases with blood groups and other
polymorphisms).
Higher incidences of infection with malaria parasites (in A individuals), plague (O), and smallpox (A)
were mentioned in the article.
Relatively recently, differential susceptibility towards Noroviruses has been reported among
individuals with different ABO phenotypes. Noroviruses are the leading cause of relatively mild
nonbacterial, acute gastroenteritis among adults and are responsible for numerous outbreaks.
Noroviruses bind type-1 H antigens, and individuals with O phenotype are more likely to be infected
by the viruses, whereas those with B phenotype have decreased risk of infection although different
strains exhibit different binding patterns and susceptibility.
Another well-characterized example is Helicobacter pylori. This bacterium can bind to the Lewis b
(Leb) oligosaccharide structure, and therefore, infects more readily individuals with certain Lewis and
ABO phenotypes. However, it should be mentioned that various strains of H. pylori also use certain
sialoglycoconjugates that are unrelated to Lewis or ABO antigens, as receptors when they bind host
cells. The routes of infection are not as simple as they were first imagined.
Appendix 27. ABO Polymorphism & Infectious Disease Susceptibility-2
ABO Polymorphism & Disease Susceptibility
Infectious Diseases (2)
In addition to the binding of infectious agents to ABH antigens on host cells, another type of interaction
also exists between ABH antigens on viruses and naturally occurring antibodies against A/B antigens.
ABH antigens may be added to the viral proteins while they are synthesized in the cells producing
those antigens. The glycolipids of membrane-encapsulated viruses may also possess ABH antigens
from the cells. Because those viruses may exhibit ABO polymorphism and the new host may contain
antibodies against A/B antigens in sera, the host ABO phenotype is likely to affect the susceptibility
towards those viruses.
The first demonstration of such interaction came with the α1,3Gal epitope and the antibody against
that oligosaccharide structure.
Retrovirus produced in mouse cells express the α1,3Gal epitope.
The GGTA1 gene which encodes the α1,3Gal transferase, an enzyme evolutionarily related with A
and B transferases, is non-functional and has become a pseudogene in Homo sapiens. Therefore,
humans cannot synthesize the α1,3Gal epitope. Instead, humans possess antibodies against this
structure. The anti-α1,3Gal epitope antibody in human sera could inactivate the retroviruses produced
by murine cells. Apparently, this seems to be one of the molecular mechanisms that inhibit the interspecies infection of the retroviruses from mouse to human. It should be noted that once infection is
established, the viruses take the phenotype of the new host, and the inhibition will no longer be
effective.
We thought of a similar scheme with the ABO phenotype of the host.
Collaborating with Dr. Hirokazu
Inoue, we produced xenotropic retroviruses from HeLa cells exhibiting different ABO phenotypes (The
ABO phenotype of the original cells was O. We transfected expression constructs encoding for human
A and B transferase and produced HeLa cells with A and B phenotypes). Those viruses were mixed
with sera from individuals with different ABO blood types, and the changes in infectivity were
monitored. We anticipated selective inhibition; however, the results showed that most viruses were
inactivated by the sera irrespectively of the ABO phenotype of the virus-producing host cells and of
the sera. Although we failed to show the inhibition of the intra-species infection of retroviruses, other
groups were successful. Using the model systems of measles viruses and HIVs, the ABO mediated
selective inhibition was demonstrated.
It is possible that the differential susceptibility to such selective forces as infectious agents may have
resulted in the observed geographical and racial differences in ABO-phenotypes frequency.
Appendix 28. ABO Polymorphism & Cancer Susceptibility
ABO Polymorphism & Cancer Susceptibility
The association between gastric cancer and blood group A was one of the first associations observed
between the ABO polymorphism and human diseases. Soon after, the association between
duodenal/gastric ulcers and group O was discovered (See the ABO polymorphism and Infectious
Diseases section for the preference binding of Helicobacter pylori to Leb). In individuals with functional
A or B genes, the Leb structure is converted to ALeb or BLeb structures by A or B transferase,
respectively. Therefore, the decreased incidence of ulcers in those individuals has been attributed to
a diminished binding of H. pylori.
Although the number of cases analyzed was smaller, a strong association with groups A and B with
pancreatic cancer was also reported. Those results were based on the comparison between the
incidences in a diseased population and its corresponding healthy population. A individuals have 25%
more chance of getting those cancers than O individuals.
Appendix 29. ABH Antigen Expression & Cancer
ABH Antigen Expression & Cancer
In addition to the differential susceptibilities depending on the ABO phenotype, altered expression of
ABH antigens in cancer has also been reported. Loss of A/B antigens was first reported in 1950s in
human gastric carcinoma, and was subsequently demonstrated to occur in the majority of squamous
and transitional cell carcinomas. Tumor cells with decreased expression of these antigens were
shown to have a higher metastatic tendency.
Life expectancy of patients with blood group A or AB
who had primary non-small-cell lung carcinomas negative for A antigen was shown to be significantly
shorter when compared to patients with A antigen-positive tumors.
Loss of A/B antigens has also been reported in prostate cancer, regardless of the histological grade
or blood type. Vowden et al. used monoclonal antibodies directed towards A, B, H and Lewis y (Ley:
Fuc α1->2 Gal β1->4 (Fuc α1->3) GlcNAc β1->) antigens and observed a loss of A/B antigen
expression in all prostate tumors tested. They also identified type 2 (core structures are Gal β1->3
GlcNAc β1-> and Gal β1->4 GlcNAc β1-> for type 1 and type 2, respectively) H and Ley antigens in
most tumors and proposed a link between type 2 structures and malignant transformation. Other
reports confirmed the augmentation of type 2 H expression in poorly differentiated prostate
adenocarcinomas, a loss of A, B, Lewis a (Lea: Gal β1->3 (Fuc α1->4) GlcNAc β1->) and Lewis b
(Leb: Fuc α1->2 Gal β1->3 (Fuc α1->4) GlcNAc β1->) expression in all grades of adenocarcinomas,
and strong expression of Ley in adenocarcinomas. In addition to Ley, sialyl Lex (sLex: NeuAc α2->3
Gal β1->4 (Fuc α1->3) GlcNAc β1->) was also found to be highly expressed in malignant prostate
tissue although it is completely absent or minimally expressed in benign secretory epithelial cells. The
appearance of Thomsen-Friedenreich antigen (T antigen: Gal β1->3 GalNAc α1->Ser/Thr) has also
been reported in a majority of prostate carcinomas.
Appendix 30. ABO & Pancreatic Cancer
ABO Polymorphism & Pancreatic Cancer
A statistically significant difference was observed in the susceptibility to stomach and pancreatic
cancers among individuals with different ABO blood groups in classic association studies. However,
those reports compared the incidences between the diseased population and the corresponding
healthy population. Because 25% difference was not so dramatic, those results were received with
some distrust in the field.
This situation has entirely changed after one paper was published in Nature Genetics in September
last year. In the paper entitled, “Genome-wide association study identifies variants in the ABO locus
associated with susceptibility to pancreatic cancer”, Amundadottir et al. genotyped 558,542 SNPs in
1,896 individuals with pancreatic cancer and 1,939 controls drawn from 12 prospective cohorts plus
one hospital-based case-control study. They also conducted a combined analysis of these groups
plus an additional 2,457 affected individuals and 2,654 controls from eight case-control studies,
adjusting for study, sex, ancestry and five principal components. They found the highest association
between SNPs and pancreatic cancer in the ABO blood group locus (SNP rs505922: combined P =
5.37 x 10(-8); multiplicative per-allele odds ratio 1.20; 95% confidence interval 1.12-1.28). As opposed
to previous targeted studies of the ABO blood groups, this genome-wide association study (GWAS)
conclusively revealed the involvement of the ABO genetic locus with pancreatic carcinogenesis.
Another group has confirmed the association between the ABO polymorphism and pancreatic cancer
(Wolpin et al. Pancreatic Cancer Risk and ABO Blood Group Alleles: Results from the Pancreatic
Cancer Cohort Consortium. Cancer Res. 2010: 70(3); 1015-1023).
Appendix 31. ABO & Diet
ABO Polymorphism & Diet
Within the gene, at the nucleotide sequence level, the differences among individuals with different
human ABO alleles are minimal with only minor substitutions and deletions/insertions. These
differences result in changes in the gene-encoded proteins: A and B glycosyltransferases and nonfunctional O proteins. The specificity and activity of the glycosyltransferases encoded by weak and
rare A and B subgroup alleles, as well as cis-AB and B(A) alleles that specify the expression of both
A and B antigens by single genes, are modified. The functional enzymes, A and B transferases, are
involved in the biosynthesis of the oligosaccharide A and B antigens, respectively.
Because these antigens are expressed on the epithelial cells of gastrointestinal tract, in addition to
red blood cells (RBCs), it is theoretically possible that these antigens may interact with carbohydraterecognizing proteins like lectins present in the diet. Because A and B antigens are carried on
glycoproteins and glycolipids on the cell surface, they may also modify the functions of those
glycoconjugates.
ABH antigens are not restricted to the humans, but they are also present in nature. Therefore, it is
possible that A/B antigens in the diet may also interact, within the human body, with naturally occurring
antibodies against those antigens and/or with lymphocytes that carry those antibodies, in addition to
the carbohydrate-binding proteins.
Appendix 32. ABO Blood Type Diets
Opinions on “Blood Type Diets”
In his book entitled, “Eat Right 4 Your Type”, Peter D’Adamo claimed that human ABO blood type is
the most important factor in determining a healthy diet, and proposed distinctive diets for individuals
with different ABO blood groups. He reasoned that the reactions to lectins (carbohydrate-binding
proteins) present in food depend on the individual ABO blood group and that the food containing
incompatible and harmful lectins would better be avoided in order to minimize toxic reactions caused
by lectin-A/B antigen interactions. This theory met with much skepticism and criticism from the medical
and scientific community. Major criticisms have centered on the lack of scientific evidence and
discrepancies with scientific norms. For example, lectins that possess high affinity to a particular ABO
type are far less common in food, except for some beans. And actually no data were presented to
correlate the kinds of lectins present in the diet and their ABO specificity.
Even if his theory is correct, it is impossible to propose right “Blood type diets” without knowing which
lectins (including those with low affinity) are contained or absent in which foods. Unless the presence
of blood group-specific lectins is demonstrated in specific diets, the promotion of the “Blood type diets”
is wrong. It may potentially harm people by proposing diets that may contain some lectins to cause
problematic interactions and/or by suggesting them to avoid some foods of high nutrient value
because of his mistaken assignments.
His belief that O, A, B, and AB blood types originated 30,000, 20,000, 10,000, and 1,000 years ago,
respectively, does not fit with the current theory of the evolution of the ABO gene, either.
Although his best seller has contributed much to enhance the people’s interest in the ABO blood
groups, Dr. D’Adamo’s proposed “Blood type diets” have nothing to do with the ABO blood types.
Appendix 33. ABH Antigens in Neurobiology
ABH Antigens in Neurobiology
In humans, ABH antigens are expressed in primary sensory neurons of the posterior root ganglia in
the nervous system. However, little is known about the roles these antigens may play. The situation
is similar with mice. However, there have been some advances.
We have previously shown that mice have an ABO gene (cis-AB gene) that encodes an enzyme with
both A and B transferase activity using heterologous transfection experiment in the human cancer cell
line HeLa. However, in vivo, A antigen is found, primarily in the gastrointestinal tract, and B antigen is
rarely detected. H antigen is expressed by primary sensory neurons in both the main and accessory
olfactory systems while A antigen is expressed by a subset of vomeronasal neurons in the developing
accessory olfactory system.
St. John et al. have used both loss-of-function and gain-of-function approaches to manipulate
expression of these carbohydrates in the olfactory system and demonstrated the following: (1). In null
mutant mice lacking the α1,2-fucosyltransferase (FUT1) and the H antigen, there was a delay in the
maturation of the main olfactory bulb glomerular layer (2). Ubiquitous expression of A antigen on
olfactory axons in a gain-of function transgenic mice caused mis-routing of axons in the main olfactory
bulb glomerular layer and led to exuberant growth of vomeronasal axons in the accessory olfactory
bulb.
Although not perfectly, these results provided the first in vivo evidence suggesting a role for specific
cell surface carbohydrates in the development of the olfactory nerve connectivity.
Appendix 34. ABO & Personality
ABO Polymorphism & Personality
The idea that the ABO blood type is partially involved in the determination of personality is still quite
common. This belief is especially popular in Japan and Taiwan. A series of books written by Masahiko
Nomi seem to have contributed, to some degree, to the popularity of this theory. The books depicted
numerous anecdotal examples, but the statistical analyses were based on subjective data rather than
objective one.
Because of this lack of objectivity, I do not think that any association between ABO polymorphism and
personality has been really demonstrated. All the claims currently made on this regard seem to be
groundless. Or at least they do not seem to withstand scientific evaluation.
ABH antigens are expressed in the primary sensory neurons of the posterior root ganglia in the
nervous system. Although there is no direct connection between the sensory neurons and personality,
it is still possible that the ABO polymorphism may affect the response of those cells. Apart from
neurons, the polymorphic expression of the ABH antigens on other types of cells, including RBCs in
circulation, some cells in the digestive tract, some cells in respiratory, endocrine, urinary, and genital
organs may indirectly affect the personality.
ABO gene is one of 25,000-plus genes contained in the human genome. But because of the
abundance and wide distribution of ABH antigens, I will not be so surprised if in the future, the
currently fashionable genome-wide association studies (GWAS) find an association between certain
definable personality traits and ABO SNPs.
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