ABSTRACT
Histo-blood group A transferase produces A antigens and
transfers GalNAc to the acceptor substrate, H structures of glycolipids
and glycoproteins. B transferase transfers galactose in place of GalNAc
to the same acceptor substrate to synthesize B antigens. We have
previously identified four amino acid substitutions between human A and
B transferases. Out of these four, substitutions at the last two
positions (codons 266 and 268) were found to be crucial for the
different donor nucleotide-sugar specificities between A and B
transferases as analyzed by gene transfer of chimeric A-B transferase
genes.
In the present study, we have in vitro mutagenized
codon 268 of these two transferase cDNA expression constructs (glycine
and alanine in A and B transferases, respectively) and produced
substitution constructs with every possible amino acid residue at this
position. We examined the activity and specificity of each construct by
gene transfer followed by immunodetection of A and B antigens and in vitro enzymatic assay. Amino acid substitution constructs
on the A transferase backbone with alanine, serine, and cysteine
expressed enzymes with A and B transferase activities. Weak A activity
was detected with histidine and phenylalanine constructs while weak B
activity was detected with asparagine and threonine constructs. All the
other amino acid substitutions at codon 268 on the A transferase
backbone showed neither A nor B activity. The glycine construct on the
B transferase backbone expressed both A and B transferase activities.
Some substitution constructs on the B transferase backbone maintained B
activity while some other substitutions abolished the activity.
These results show that the side chain of the amino acid residue at
268 of the human A and B transferases is responsible for determining
both activity and nucleotide-sugar donor substrate specificity and
strongly suggest its direct involvement in the recognition of and
binding to the sugar moiety of the nucleotide-sugars.
Glycoproteins and glycolipids are essential components of
cellular membranes. Carbohydrate structures of these molecules are
complex and exhibit great variety, and they are synthesized through a
series of reactions catalyzed by the enzymes called
glycosyltransferases(1, 2) . The human histo-blood
group ABO system offers one of the best systems to study the structural
basis of specificity and activity of glycosyltransferases(3) .
The functional alleles at the ABO locus encode glycosyltransferases to
catalyze the final step of reactions to synthesize the carbohydrate
antigens A and B(4) . A
alleles encode an enzyme
(A
transferase) to transfer N-acetyl-D-galactosamine (or GalNAc for short) from
the donor substrate, UDP-GalNAc, to the acceptor substrate H structures
of glycoproteins and glycolipids. B alleles encode another enzyme (B
transferase) to transfer a different sugar, D-galactose from
UDP-galactose, to the same acceptor substrate. Therefore, comparative
structural analysis is needed to delineate the structural basis of the
different specificities of A and B transferases. O alleles are
incapable of producing functional enzymes, and the substrates remain
unchanged. In addition to these three major alleles, there are many
other minor subtypes. Although they are rare in the population,
glycosyltransferases coded by these subtypic alleles offer clues about
the structural basis of the enzymatic activity of glycosyltransferases.
We have cloned the cDNA for the human histo-blood group A
transferase (5) and used this cDNA to screen cDNA libraries
made with RNA from human cell lines showing different ABO phenotypes.
We then co-related the differences in the nucleotide sequences with
each of the three major (A
, B, and O) alleles(6) .
Four amino acid substitutions (RGLG and GSMA at codons 176, 235, 266,
and 268 in A and B transferases, respectively) were identified, which
discriminate between A and B transferases. O alleles were found to
possess a single nucleotide deletion relatively close to the amino
terminus of the coding sequence of the enzyme. We then analyzed A
alleles, one of the A subtypes, and identified another single
nucleotide deletion, which, different from the single nucleotide
deletion of O alleles, is located at the carboxyl terminus of the A
transferase coding sequence(7) . This deletion changes the
frame of codons and results in a transferase (A
transferase) with an additional 21 amino acids at the carboxyl
terminus. The presence of this domain was then proven to diminish the
activity and confer restriction on the acceptor substrate usage of the
enzyme. Sequence analysis of the alleles responsible for various minor
subtypes(8, 9) and two interesting phenotypes, cis-AB (10) and B(A)(9) , also revealed other
mutations in the gene. We also determined the partial nucleotide
sequences of ABO genes from some species of primates (11) .
Among the four amino acid substitutions that discriminate human A and B
transferases, amino acid residues at only the third and the fourth
positions were found to be conserved in the primate ABO genes. All the
information from these sequence analyses, in combination with
functional assays of A and B transferases and their
chimeras(12) , indicate the importance of amino acid residues
at 266 and 268 of the human A and B transferases for the activity and
the different donor substrate specificities of these two enzymes.
We
have recently identified a second type of O allele that is devoid of
the single nucleotide deletion found in all other O alleles previously
analyzed and instead possesses two amino acid substitutions, one at
codon 176 (arginine to glycine) and the other at codon 268 (glycine to
arginine) of A transferase(13) . The presence of this O allele
in the Danish population has been reported(14) . DNA
transfection experiments have shown that the introduction of these two
missense mutations to a functional A transferase expression construct
completely abolished the enzymatic activity. Because the arginine to
glycine substitution at codon 176 was incapable of changing activity or
specificity of the enzyme(12) , we speculated that the glycine
to arginine substitution at codon 268 was responsible for the
inactivation of the transferase. Sequence comparison of A and B
transferases with bovine (15) and murine (16)
-1,3-galactosyltransferases confirmed the importance
of codon 268 (rather than codon 266) of the human A and B transferases
for different donor nucleotide-sugar specificities.
In order to test
more directly this hypothesis and learn more about the structural
specificities of these enzymes, we produced amino acid substitution
constructs with every possible amino acid residue at position 268 in
both A and B transferases. We then analyzed the effect of the amino
acid residue at codon 268 on the nucleotide-sugar donor substrate
specificity and enzymatic activity of A and B transferases.
EXPERIMENTAL PROCEDURES
Materials
The reagents for PCR (
)were
purchased from Perkin-Elmer, and reagents for DNA sequencing were from
U. S. Biochemical Corp. (Cleveland, OH). Radioactive
[
P]dATP was from DuPont NEN, and
[
C]UDP-galactose and
[
C]UDP-GalNAc were from Amersham Life Science
(Arlington Heights, IL). Restriction enzymes and LipofectAMINE
reagent were from Life Technologies, Inc. Frozen competent
bacteria XL1-Blue strain of Escherichia coli was from
Stratagene (La Jolla, CA). Anti-A and anti-B mixtures of murine
monoclonal antibodies were from Ortho Diagnostic Systems Inc.
(Piscataway, NJ), and fluorescein isothiocyanate-conjugated goat
anti-mouse immunoglobulin was from Sigma.
-Galactosidase assay kit
was from Promega (Madison, WI). 2`-Fucosyllactose, UDP-galactose,
UDP-GalNAc, and 5-bromo-4-chloro-3-indolyl
-D-galactoside
(X-gal) were from Sigma, and Poly-Prep prefilled AG1-X8 resin
chromatography columns were from Bio-Rad. Degenerate
oligodeoxynucleotides FY-78, FY-81, and PDM-1 to -6 were custom
synthesized at BioSynthesis Inc. (Denton, TX). The nucleotide sequences
of these oligodeoxynucleotide primers are as follows: FY-78,
CCGGATCCGTGTGATTTGAGGTGGGGAC; FY-81,
CGGAATTCA(A/T)G(T/C)ACTTCATGGT(G/T)GGCCA; PDM-1,
ATTTCTACTACCTGGGGNNNTTCTTCGGGGGGTCGGT; PDM-2,
ACCGACCCCCCGAAGAANNNCCCCAGGTAGTAGAAAT; PDM-3,
ATTTCTACTACATGGGGNNNTTCTTCGGGGGGTCGGT; PDM-4,
ACCGACCCCCCGAAGAANNNCCCCATGTAGTAGAAAT; PDM-5,
ACCCCCCGAAGAACATCCCCAGGTAGTA; PDM-6, ACCCCCCGAAGAA(C/T)ATCCCCATGTAGTA.
The BamHI site of FY-78 and the EcoRI site of FY-81
are artificial. The capitalized underlined letter N denotes a mixture
of four nucleotides (GATC) at the position.
Construction of Amino Acid Substitution
Constructs
We employed polymerase chain reaction with degenerate
oligodeoxynucleotides to introduce mutations at codon 268 of human A
and B transferases (Fig. 1). Two DNA fragments were first
amplified from the template plasmid pAAAA (or pBBBB) DNA(12) ,
with two sets of oligodeoxynucleotide primers (FY-81 and PDM-2 (or -4)
or FY-78 and PDM-1 (or -3), respectively). Oligo primers, PDM-1 and -2
(and PDM-3 and -4), possess degenerate nucleotide sequences(NNN) at
codon 268 to represent all the possible 64 combinations. The two
primers, PDM-1 and -2 (and PDM-3 and -4), are complementary to each
other except at codon 268. Supplemented with the original plasmid DNA,
the two amplified fragments overlapping one another at the primer
sequence were used as templates to amplify the fragment bordered by the
outer primers, FY-78 and -81. Oligo primer FY-81 contained a degenerate
sequence, but the DNA portion containing this sequence was removed from
the insert by SstII and BamHI double digestion before
ligation with the remaining portion of A (or B) transferase expression
construct. The ligated DNA was used to transform the XL1-Blue strain of E. coli. Plasmid DNA was prepared from individual
transformants and analyzed for the presence of inserts, and DNA
containing the insert was denatured and used as a template for DNA
sequencing reactions by Sanger's dideoxy chain termination method
using the Sequenase
sequencing kit. The nucleotide
sequence of codon 268 and its surrounding region (from nucleotide 695
to nucleotide 885) of 381 and 259 clones of A and B transferase amino
acid substitution constructs, respectively, was determined for
individual clones. Multiple clones with the correct sequence over the
region were obtained for each amino acid substitution construct except
for methionine constructs. In order to obtain methionine substitution
constructs at codon 268, we used primer PDM-5 (or -6) in place of PDM-2
(or -4). Primer PDM-5 has a methionine codon (ATG) at amino acid 268 in
the A transferase sequence background whereas PDM-6 has a mixture of
methionine and isoleucine codons (AT(G/A)) at this position in the B
transferase sequence background. We used degenerate primer PDM-6
because the number of isoleucine constructs was small when this
oligonucleotide primer was designed. Further DNA sequencing was
performed over the entire coding region, which was amplified by PCR
(from the SstII site to the termination codon between
nucleotides 470 and 1065) in order to exclude the clones with any
additional unexpected mutation(s). We have obtained at least one such
clone for each amino acid substitution construct.
Figure 1:
Locations of primers. The
locations of primers are schematically shown. Coding regions of the
human A and B transferase cDNAs are shown by open bars. The
location of the remaining intron in the construct is also indicated
(not on the scale). The A residue and the methionine residue of the
initiation codon of A and B transferases are numbered 1. There
are four amino acid (aa) substitutions between A and B
transferases, and their positions are indicated
(Arg
-Gly
-Leu
-Gly
in A and
Gly
-Ser
-Met
-Ala
in B). Two overlapping fragments were PCR-amplified using two
sets of oligodeoxynucleotide primers (FY-81 and PDM-2 (or -4) and FY-78
and PDM-1 (or -3)) and template plasmid (pAAAA or pBBBB). The entire
long fragment was then amplified with the two outermost primers (FY-78
and FY-81), cleaved with SstII and BamHI, and ligated
with the vector portion of similarly digested original plasmid. (There
is a BamHI site in the vector pSG-5, which locates downstream
of the A and B transferase cDNA inserts.) After DNA transformation,
insert-containing clones were identified, and the partial nucleotide (nt) sequence around the codon 268 was determined. Those
clones without additional mutations in the sequenced region were
further sequenced to their entirety over the coding region amplified by
PCR (from the SstII site to the termination codon) and used
for DNA transfection experiments.
DNA Transfection, Immunostaining, FACS Analysis, and
Enzymatic Assays
Two cell lines (HeLa and COS 1) were used as
recipients of DNA transfection experiments. LipofectAMINE
reagent (17) was used as per the manufacturer's
instructions. DNA from a single representative clone (whose coding
sequence was confirmed to be correct over the PCR-amplified region)
from each amino acid substitution was co-transfected with DNA from
pSV-
-galactosidase control vector. Two days after transfection,
HeLa cells were harvested and fixed. Portions of cells were
immunostained with a mixture of murine anti-A or anti-B monoclonal
antibodies and secondarily with fluorescein isothiocyanate-conjugated
goat anti-mouse immunoglobulin antibody, and they were subjected to
FACS analysis. The percentages of cells stained for
-galactosidase
activity in situ(18) were used to normalize the
different transfection efficiencies among constructs. DNA-transfected
COS 1 cells were harvested and lysed, and the supernatant was used for
A and B transferase assays in vitro. A and B transferase
activities were analyzed by the transfer of the radioactivity from
[
C]UDP-GalNAc and UDP-galactose to the acceptor
substrate, 2`-fucosyllactose, respectively. The reaction was performed
in a mixture of 100 mM sodium cacodylate (pH 6.5), 5 mM ATP (pH 6.5), 20 mM MnCl
, 0.1 mM UDP-GalNAc or UDP-galactose (containing
C-labeled
UDP-GalNAc or UDP-galactose, respectively), and 0.5 mM 2`-fucosyllactose. The reaction mixture containing the cell
extract was incubated at 37 °C for 6 and 2.5 h for A and B
transferase assays, respectively. Incorporation of the radioactivity
was found nearly proportional to the incubation time for the original A
and B transferase under these conditions. The reaction without the
acceptor substrate was used as a negative control. Radioactivity was
measured with a scintillation counter after separation of enzymatic
reaction products from the donor nucleotide-sugar substrates through
the AG1-X8 anion exchange column chromatography. The supernatant was
also used for
-galactosidase enzymatic assay. Activity was
determined for each sample by measuring hydrolysis of o-nitrophenyl-
-D-galactopyranoside, a substrate
for
-galactosidase, and the resulting value was used to normalize
DNA transfection efficiency.
RESULTS
Specificity and Activity of A Transferase Amino Acid
Substitution Constructs
Two cell lines were used as recipients
of DNA transfection. HeLa cells express type H antigens on the cell
surface and have been used successfully in our previous studies as
recipients of DNA transfection of A and B transferase expression
constructs and their derivatives(7, 12, 13) .
The specificity and activity of each amino acid substitution construct
were analyzed by immunodetecting A and B antigens, which appeared on
the surface of DNA-transfected cells by FACS. DNA from a single
representative clone, whose nucleotide sequence was confirmed to be
correct over the PCR-amplified coding sequence, from each amino acid
substitution construct was used for DNA transfection with
LipofectAMINE
. A construct containing a cDNA from the O
allele in pSG-5 vector in the antisense orientation was used as a
negative control, and the intensity of fluorescence of HeLa cells
transfected with this DNA was measured and the borderlines to
discriminate FACS positive cells from negative ones were placed
accordingly. COS 1 cells, which produce T antigens to trigger high
expression of genes under control of the SV 40 promotor sequence, were
also used as recipients of DNA transfection to obtain the maximum
expression of the constructs made in the vector pSG-5. The enzymatic
activities of A and B transferases were measured in vitro with
extracts from transfected COS 1 cells.The results of immunoassays
of HeLa cells and those of enzymatic assays of COS 1 cells, which were
transfected with DNA from A transferase amino acid substitution
constructs, were normalized for the different efficiencies of DNA
transfection and are shown in Table 1. The median intensities of
fluorescence among the positively stained cells are also indicated in
parentheses. Based on all the experimental results including the data
from the pilot experiments (data not shown), A and B transferase
activities of each amino acid substitution construct were determined
and shown in the two far right columns.
The original A transferase
glycine construct expressed very strong A transferase activity as
expected. An A transferase alanine construct expressed strong A
transferase activity, which was comparable with that of the glycine
construct, but this alanine construct expressed weak B transferase
activity as well. Two A transferase substitution constructs (serine and
cysteine) expressed moderately strong A and B activities. Histidine and
phenylalanine constructs showed very weak A activity, and asparagine
and threonine constructs showed very weak B activity. Several A
transferase constructs with other amino acids at codon 268 showed no
activity of A or B transferase.
Specificity and Activity of B Transferase Amino Acid
Substitution Constructs
The results of DNA transfection
experiments with B transferase amino acid substitution constructs are
shown in Table 2. The original B transferase alanine construct
expressed only B activity in FACS analysis; however, very weak A
transferase activity was measured by in vitro enzymatic assay.
One B transferase glycine construct showed strong activities of both A
and B transferases. Two B transferase substitution constructs (serine
and cysteine) expressed strong B transferase activity comparable with
that of the original alanine construct. Two constructs (asparagine and
threonine) showed strong B activity and five (aspartic acid, histidine,
leucine, proline, and valine) constructs expressed moderate B activity.
Two (glutamine and phenylalanine) and one (methionine) constructs
expressed weak and very weak B activity, respectively.
DISCUSSION
Comparison of Two Detection Methods (Immunological
Assays of A and B Antigens on the Surface of HeLa Cells and Enzymatic
Assays of A and B Transferases in COS 1 Cell Extracts)
It is
logical to assume that the detection of A and B antigens (secondary
enzymatic reaction products) is more sensitive than that of enzymes
(primary gene product) if the enzymes are translocated to the
appropriate locus (Golgi apparatus) and functional in vivo. As
shown in Table 1and Table 2, sensitivity was actually much
higher with the detection of A and B antigens than with the in
vitro enzymatic assay. The median intensities of fluorescence of
positively stained cells among different constructs seem similar,
suggesting that even small amounts of enzymatic activity generated
significant numbers of cell surface A and/or B antigenic determinants
very efficiently. We used only single in vitro enzymatic assay
conditions. However, under these conditions, we were able to detect
activity even less than 1% of the original A and B transferase
constructs using extracts from COS 1 cells transfected with each amino
acid substitution construct. For example, A transferase activity of A
transferase serine substitution construct has 0.4% of that of the
original A transferase glycine construct. COS 1 cells possessed low
activities of A and B transferases, and small amounts of radioactivity
incorporated into the reaction products (high background level) made it
difficult to detect very weak activities of some substitution
constructs. However, it is true that antigen-positive HeLa cells were
also not detectable with those constructs by immunological means when
the transfection efficiency was low. The percentages of A and B antigen
expressing HeLa cells seemed proportional to the enzymatic activities
when the expression of the enzymes was low (data not shown), whereas at
higher expression they are linear but not proportional ( Table 1and Table 2). This may be due to the saturation of A
and B antigens on the cell surface when large numbers of enzymes are
produced in vivo. We found one exception where no enzymatic
reaction products were detected on HeLa cells, but A transferase
enzymatic activity was measured in vitro in the COS 1 cell
extract (B transferase original alanine construct). This result may not
be real because the A transferase activity detected in vitro was so small (0.3% of that of original A transferase); however, a
similar finding was previously reported using sera from blood group B
individuals as an enzyme source(19) .
An A Transferase Alanine Construct Expressed Strong A and
Weak B Transferase Activities
We have previously identified two
missense mutations in two cases of cis-AB alleles responsible
for the cis inheritance of both A and B transferase activities
by a single gene on a single chromosome(10) . One of these
mutations was found at the last position of the four amino acid
substitutions that discriminate the human A and B transferases. cis-AB alleles contained the B-specific alanine at codon 268
in the A transferase backbone. Therefore, the A transferase alanine
substitution construct corresponds exactly to previously published
expression construct pAAAB. DNA transfection of this A transferase
alanine construct has yielded a result showing weak B transferase
activity, which is different from the previous result with
pAAAB(12) . The repeated analysis of the pAAAB indicates that
discrepancy may be ascribed to antibody specificity issues. The same
explanation can be applied to pBABB, another chimeric construct
representing B(A) allele(9) , which was previously found not to
express A activity(12) . Considering the fact that B(A) was
initially identified with certain murine anti-A monoclonal
antibody(20) , different anti-A antibodies may detect A
antigens on HeLa cells transfected with the pBABB construct.
Effects of Amino Acid Substitutions at Codon 268 on
Specificity and Activity of A Transferases
Clearly positive A
transferase activity was observed with six amino acid substitution
constructs with the A transferase backbone. Those amino acid residues
are glycine, alanine, serine, cysteine, histidine, and phenylalanine,
and activity decreases in that order. Apparently, the strength of
activity is reciprocally proportional to the size of side chain. The
side groups of amino acid residues other than four (glycine, alanine,
serine, and cysteine) may be too bulky to accommodate the GalNAc
portion of the UDP-GalNAc nucleotide-sugar into the recognition and
interacting site of A transferase. It is difficult to explain weak A
activity of histidine and phenylalanine constructs only by the size of
the side group; however, these amino acid residues may have special
characteristics considering the fact that these amino acid substitution
constructs on B transferase backbone also showed moderate B transferase
activity, as will be discussed. B transferase activity was observed
with serine, cysteine, alanine, asparagine, and threonine (all with
small side chains) at codon 268 in A transferase constructs. The side
chain of glycine (hydrogen) may be big enough to interact with GalNAc
but too small to interact with galactose.
Effects of Amino Acid Substitutions at Codon 268 on
Specificity and Activity of B Transferases
Strong A transferase
activity was detected only in the glycine construct with the B
transferase backbone. The methionine at codon 266 of B transferase
constructs is bigger than the leucine residue of A transferase
constructs, and this bigger size may compensate the small hydrogen side
chain of glycine at codon 268 and enable the B transferase glycine
mutant to interact with and transfer both GalNAc and galactose. Strong
B activity was observed with six B transferase constructs (alanine,
glycine, serine, cysteine, asparagine, and threonine). In addition,
five (aspartic acid, histidine, leucine, proline, and valine), two
(glutamine and phenylalanine), and one (methionine) constructs showed
moderate, weak, and very weak B activity, respectively. Comparison of B
antigen-positive cell percentages among constructs with a neutral polar
amino acid at codon 268 (glycine > serine > threonine >
tyrosine = 0) indicates the importance of the size of side chain
as shown above with A transferase substitution mutants. Comparison
among four amino acid substitutions (asparagine > aspartic acid >
glutamine > glutamic acid = 0) suggests that the presence of
an acidic side chain decreased enzymatic activity. Not only the size
but also the basic charge of the side groups may be responsible for the
loss of activity in arginine and lysine constructs. However, the
construct with histidine, a basic amino acid with an imidazole group,
showed moderate B activity. Comparison among constructs with neutral
hydrophobic side chains (alanine > valine
leucine >
isoleucine = tryptophan = 0) shows again the importance
of the size of the side chain for the strength of activity. The
phenylalanine construct, however, showed weak B transferase activity.
The construct with a proline residue, in which the nitrogen atom of the
amino group is incorporated into a ring, introducing a bend in a
peptide, still possessed B activity. In addition, more amino acid
substitutions were functional in B transferase backbone than those in A
transferase backbone. This implies that the amino acid residues at 266
and possibly 267 as well are also important in both the activity and
the specificity of the transferases and that the size of the side chain
at codon 268 is not the only issue. We hope that three-dimensional
structural analysis by x-ray crystallography and molecular modeling of
these transferases will clarify these ambiguities in the future.
O Alleles with Amino Acid Substitution Other Than
Arginine at Codon 268?
We have previously identified a second
type of O allele, which contains two amino acid substitutions
(arginine-to-glycine at 176 and glycine-to-arginine at
268)(13) . The A transferase arginine construct showed no
activity of A or B transferase suggesting that the glycine-to-arginine
substitution at codon 268 is sufficient alone to abolish A transferase
activity in the protein coded by this rare type of O allele. Single
nucleotide substitutions at the first and the second positions of codon
268 (nucleotides 802 and 803) of the A transferase coding sequence
result in five amino acid substitutions at codon 268 (from GGG: glycine
to GAG: glutamic acid, GTG: valine, GCG: alanine, AGG and CGG:
arginine, or TGG: tryptophan). Out of these five, alanine was the only
substitution that did not spoil the enzymatic activity as shown in Table 1. Similarly, single nucleotide substitutions at those
positions of the B transferase coding sequence result in six amino acid
substitutions (from GCG: alanine to ACG: threonine, TCG: serine, CCG:
proline, GGG: glycine, GAG: glutamic acid, or GTG: valine). As shown in Table 2, B transferase alanine to glycine substitution resulted
in an enzyme with both A and B transferase activities while the
glutamic acid substitution abolished the B transferase activity. All
the other amino acid substitutions (threonine, serine, proline, and
valine) did not abolish but weakened B transferase activity.In all,
five single base substitutions at codon 268 in the A transferase
backbone and one in the B transferase backbone abolish the activity and
result in phenotypically O alleles. Of these six possible mutations
only an arginine substitution at codon 268 with the A transferase
backbone has been discovered in O alleles in the human population so
far(13, 14) , in addition to the single nucleotide
deletion that distinguishes most O alleles(6) . Our present
results, however, suggest the possible existence of O alleles with
other amino acid residues at codon 268 in the A or B transferase
backbone. ABO genotyping has become
popular(6, 14, 21, 22, 23, 24) ;
however, the presence of O alleles without the single nucleotide
deletion (frameshift mutation) and possibly of A and B alleles with an
amino acid residue at codon 268 other than glycine and alanine,
respectively, may lead to unexpected conflicting results between ABO
phenotypes and genotypes. Therefore, we must bear in mind that
genotyping based on partial sequence information may not be 100%
accurate.