(Received for publication, September 26, 1995; and in revised form, January 2, 1996)
From the
The Lewis histo-blood group system comprises two major antigens,
Lewis a and Lewis b. The Lewis b antigen is a product of two
fucosyltransferases, the (1,3/1,4)fucosyltransferase (Lewis
enzyme; Fuc-TIII) encoded by the Lewis gene and an
(1,2)fucosyltransferase which is not required for synthesis of
Lewis a antigen. An enzyme responsible for secreting ABH antigens into
body secretions (secretor enzyme) is also one of
(1,2)fucosyltransferases. A candidate gene encoding secretor
enzyme Sec2 gene was recently cloned by Rouquier, S., Lowe, J.
B., Kelly, R. J., Fertitta, A. L., Lennon, G. G., and Giorgi, D.
((1995) J. Biol. Chem. 270, 4632-4639) and Kelly, R. J.,
Rouquier, S., Giorgi, D., Lennon, G. G., and Lowe, J. B. ((1995) J.
Biol. Chem. 270, 4640-4649) who demonstrated a G428A
nonsense mutation (Trp
to terminal codon) in Sec2 of nonsecretors. However, the G428A nonsense mutation discovered
in the Sec2 gene of nonsecretors in an ethnic group other than
Japanese was not found in any of 45 Japanese nonsecretors, whereas one
Filipino who had been erroneously registered as a Japanese possessed
the G428A mutation heterozygously.
In order to explore the Sec2 gene of a Japanese population, we performed a molecular genetic
analysis of the Sec2 gene on 226 Japanese individuals, 21 in a
family study and 205 in a random sampling study. We discovered two
novel mutations in the Sec2 gene, an A385T missense mutation
(Ile to Phe) that results in inactivation of Sec2-encoded
(1,2)fucosyltransferase and a C357T silent
mutation which is irrelevant to amino acid substitution, in Japanese
nonsecretors.
The analysis of Japanese individuals using the polymerase chain reaction-restriction fragment length polymorphism method found three alleles in the Sec2 gene, the first having no mutation, the second having a C357T mutation, and the third having both C357T and A385T mutations, which we designated as Se1, Se2, and sej, respectively. Among 226 Japanese individuals, 40 having a Le(a+b-) phenotype and 5 having a Le(a-b-) nonsecretor phenotype were homozygous for sej/sej, whereas 149 having a Le(a-b+) phenotype and 32 having a Le(a-b-)-secretor phenotype possessed at least one Se1 or Se2.
The frequencies of
occurrence of Se1, Se2, and sej among 410
alleles examined in a random sample of 205 Japanese individuals were
15, 46, and 39%, respectively, indicating a rather wide distribution of
the sej allele in the Japanese population. The results show
that the Sec2 gene really encodes the secretor enzyme
(1,2)fucosyltransferase and indicate that a ethnic group-specific
nonsense or missense point mutation in the Sec2 gene
determines nonsecretor status. The phylogenic aspect and biological
significance of the Se and Le genes are discussed.
The biosynthetic pathways of the Lewis histo-blood group system
in correlation with ABH antigen synthesis shown in Fig. 1are
considered to function in the epithelial cells of digestive organs,
although some of the pathways involved have not yet clearly been
proven. Individuals are divided by their erythrocyte Lewis antigen
phenotypes into three types, Le(a+b-) which has Le(
)antigen but not Le
antigen,
Le(a-b+) which has Le
but not Le
,
and Le(a-b-) having neither Le
nor
Le
. Besides these three phenotypes, Le(a+b+)
which possesses both Le
and Le
, has
occasionally been found in some ethnic groups(1) .
Figure 1: Biosynthetic pathways involved in synthesis of Lewis blood group antigens and secretion of ABH antigens into saliva.
Based on
the biosynthetic pathways in Fig. 1, both antigens, Le and Le
require involvement of the Lewis enzyme, that
has
(1,4)fucosyltransferase activity, for their synthesis. It was
verified by our and others' previous
studies(2, 3, 4) that the Lewis gene (Le gene) encoding the Lewis enzyme is the Fuc-TIII gene (5) since the FucT-III gene of
Le(a-b-) individuals was found to be inactivated by
missense mutations(2, 3, 4) . Le
and Le
antigens are thought to be synthesized in
digestive organ epithelial cells and to be secreted into blood and body
secretions such as saliva(6, 7) . Red blood cells
adsorb the glycolipids carrying Lewis antigens in plasma(8) ,
and their phenotypes are typed by a hemagglutination test. ABH antigens
of the ABO blood group system are synthesized not only in digestive
organ epithelial cells but also in red blood cell precursor
cells(9) .
Individuals have been divided into two groups,
secretors and nonsecretors, depending on the presence and absence of
ABH antigens in their saliva, respectively. The ABH antigens of
erythrocytes are the product of H gene-encoded enzyme which
synthesizes both H type-1 and H type-2 structures by
(1,2)fucosyltransferase activity. Bombay individuals who lack H
antigens of erythrocytes were shown to be mutants whose H gene
is completely inactivated by the nonsense mutation(10) . Some
para-Bombay individuals who lack H antigens of erythrocytes, but can
secrete ABH antigens into saliva (secretor), were also found to be H gene mutants, with the nonsense mutation causing complete
inactivation of the H enzyme(10) . Some para-Bombay
individual's ability to secrete ABH antigen into saliva was
therefore thought to be determined not by the H enzyme, but by the
other
(1,2)fucosyltransferase that should be encoded by the
secretor gene (Se gene).
The Le(a-b+) phenotype
of erythrocytes is usually in accord with secretor status in saliva,
whereas the Le(a+b-) phenotype is in accord with nonsecretor
status. Although inconsistency between the Le(a-b+) and
secretor phenotype or between the Le(a+b-) and nonsecretor
phenotype is occasionally observed, this could be due to an ambiguous
hemagglutination of erythrocytes in the test or a failure in detection
of ABH antigens in saliva. If the pathways in Fig. 1actually
operate in digestive organ epithelial cells, individuals lacking the Se
enzyme cannot synthesize the H type-1 structure that is a precursor of
A, B, and Le antigens, and this should reveal their
nonsecretor status. Also according to the pathways in Fig. 1,
the erythrocyte phenotype of individuals who lack the Se enzyme should
be Le(a+b-) or Le(a-b-), and not
Le(a-b+).
A candidate for the Se gene, termed Sec2, having a highly homologous sequence to the H gene(11) , has recently been cloned from a
rat(12) , human(13, 14) , and
rabbit(15) . The recombinant enzyme directed by the Sec2 gene apparently had (1,2)fucosyltransferase activity,
transferring fucose preferentially to type-1 chain acceptor
oligosaccharides rather than type-2 chain acceptors. Substrate
specificities of the Sec2-directed enzyme (12, 13, 14, 15) were also very
similar to those of the Se enzyme previously purified by biochemical
methods(16) . It was proven that the Sec2 gene is the Se gene by these molecular analyses(13, 14) .
Due to recent successes in cloning(13) , three homologous
genes, H, Sec1, and Sec2, were found to be
in close physical linkage to each other within 100 kb on human
chromosome 19. One of them, termed the Sec1 gene, seemed to be
a pseudogene in which the open reading frame is interrupted by
translational frameshifts and termination codons(14) . The
three genes, H, Sec1, and Sec2, form a gene
cluster and are mapped on 19q13.3(13) , suggesting that they
evolved by gene duplication, as is the case in the genes of some
members of the
(1,3)fucosyltransferase family, i.e.
Fuc-TIII, -TV, and -TVI, which cluster on the
short arm of chromosome 19p13.3(17, 18) . The human Sec2 gene, a candidate for the human Se gene, has
been analyzed by Kelly et al.(14) on a molecular
genetic basis. They found a nonsense mutation G428A which changed
Trp
into a termination codon in the Sec2 gene of
a nonsecretor individual and completely inactivated the Sec2 gene-directed enzyme. It was also demonstrated that all six
nonsecretors among 52 individuals randomly sampled had the G428A
nonsense mutation homozygously. This strongly suggests that the Sec2 gene encodes the Se enzyme(14) .
In this study, we performed a molecular genetic analysis of the Sec2 gene on 226 Japanese individuals comprising 21 individuals belonging to four pedigrees and 205 randomly sampled individuals and found the novel mutations, C357T and A385T, differing from G428A, in the Sec2 gene of the Japanese nonsecretors. The allele inactivated by a novel single missense mutation A385T, termed sej, was distributed homozygously in all Japanese nonsecretors examined in this study, whereas no G428A mutation in the Sec2 gene was found in the Japanese nonsecretors.
In this report, we present these results together with the finding that Se and Le genotypings performed in parallel distinguished three Lewis phenotypes, Le(a+b-), Le(a-b+), and Le(a-b-), as well as secretors and nonsecretors with 100% accuracy, and discuss the phylogenic aspect and biological significance of the Se and Le genes.
Figure 4: Family studies on the Sec2 gene. a, erythrocyte phenotypes, secretor status, and Sec2 genotypes determined on 21 individuals of four pedigrees are shown. Square, male; circle, female; filled symbol, homozygote of sej allele (sej/sej); half-filled symbol, heterozygote of Se and sej alleles (Se1 or Se2/sej); open symbol, homozygote of Se allele (Se1 or Se2/Se1 or Se2). Closed triangles and open circles in schematic alleles represent C357T and A385T mutations, respectively. b, electrophoresis patterns on PCR-RFLP. C357T, AseI digestion for detection of the C357T mutation; A385T, AluI digestion for detection of the A385T mutation. Control PCR-RFLPs were done using the cloned plasmid DNA having each allele as templates. +/+, PCR-RFLPs using the cloned relevant mutant allele into pBS as a template; -/-, PCR-RFLPs using the cloned allele having no relevant mutation as a template; +/-, PCR-RFLPs using the mixed templates of the cloned wild-type allele and the cloned mutant allele.
For detection of the G428A nonsense mutation, the tk3 primer was designed as a mismatched primer to create an XbaI site, TCTAGA, in the PCR product from the mutant allele having the G428A. In case of the product having the G428A mutation, the second PCR product amplified by the primer set of tk3 and tk4 was separated into two fragments, 84 and 23 bp, by XbaI digestion.
For detection of the C357T and A385T mutations in the Sec2 gene that had been discovered by complete sequencing of the Sec2 gene of YoN (a Le(a+b-) nonsecretor phenotype), the second PCRs were performed by the primer sets, tk5 and tk6, and tk7 and tk8, respectively. The tk5 primer was designed as a primer mismatched at two positions of the Sec2 gene sequence by changing C to A at position 352 and A to T at position 354. These artificially changed bases are indicated by boldface letters in the sequence of primers in Table 1. The tk5 primer created an AseI site, ATTAAT, in the second PCR product from the mutant allele having C357T. The 98-bp PCR product was cleaved into two fragments, 70 and 28 bp, by AseI digestion. For detection of the A385T, the tk7 primer was designed as a primer mismatched at two positions, 382 and 383, to create an AluI site, AGCT, in the second PCR product from the mutant allele having the A385T mutation. The PCR product with this mutation was cleaved into two fragments, 51 and 29 bp, by AluI digestion.
For detection of the three missense mutations, T59G, G508A, and T1067A, in Le genes, the same primer sets as reported in the previous study (3, 21) were used except for the detection of G508A where a new primer set, sn8 and sn9, was designed in this study to improve the PCR condition. The methods for detection of the three mutations in Le genes using these primer sets are described in detail in our previous studies(3, 21) .
The second PCR products digested with the enzymes shown in Table 1were electrophoresed in a 4% agarose (NuSieve GTG agarose, FMC Corp. BioProducts, Rockland, ME), and the separated bands were observed after ethidium bromide staining.
Figure 2: Absence of G428A mutation in Japanese nonsecretors. From left to right, 10 Japanese nonsecretors showing no G428A mutation; EA, a Filipino, showing a heterozygous pattern for G428A mutation; M, molecular size markers (bp).
We also sequenced the full-length Sec2 gene of SN, in Family 1 in Fig. 4, who has a Le(a-b-) secretor phenotype. The results revealed that three of eight clones shared the C357T mutation without any other mutation, but the other five clones had no mutation at all.
We designated the three alleles in Sec2 gene, the first having no mutation, the second having C357T alone, and the third having both C357T and A385T, as Se1, Se2, and sej, respectively. In Fig. 3, we show the schematic primary structure of the enzyme and these three alleles together with the positions of primer sets for detection of the mutations by PCR-RFLP methods to be described in the following section.
Figure 3:
Schematic diagrams of PCRs employed for Sec2 genotyping by PCR-RFLP, three Sec2 alleles
having point mutations and PCR-RFLP methods for detection of C357T and
A385T. a, the positions of four primer sets for PCRs. The tk1
and tk2 set was employed for full-length Sec2 gene
amplification. The primer sets, tk3 and tk4, tk5 and tk6, and tk7 and
tk8, were employed for detecting G428A, C357T, and A385T, respectively.
Schematic primary protein structure of the Sec2-directed enzyme: C, cytoplasmic tail; TM, transmembrane domain; CR, catalytic region. Three alleles found in the Japanese
population are Se1, Se2, and sej. The Se1 allele has the same sequence as originally defined (10) without any mutations. The Se2 allele has a
silent mutation, C357T, and the sej allele has C357T and a
missense mutation, A385T (Ile to Phe). b,
schematic diagram of PCR-RFLP methods for detection of point mutations,
C357T (
) and A385T (
). For detection of the C357T mutation,
the 98-bp PCR products from Se2 and sej alleles using
the primer set of tk5 and tk6 are cleaved into two fragments by AseI digestion, whereas the product from the Se1 allele is not cleaved by AseI digestion. For detection of
A385T mutation, the 80-bp product from the sej allele using
the primer set of tk7 and tk8 is cleaved into two fragments by AluI digestion, whereas the products from Se1 and Se2 alleles are not cleaved.
The primer sets mismatched to the original sequence of the Sec2 gene for detection of the two mutations, C357T and A385T, were designed for PCR-RFLP as described under ``Materials and Methods.'' As seen in the electrophoresed patterns of PCR-RFLP in Fig. 4, the alleles having the C357T mutation, Se2 and sej, were separated into two fragments, 70 and 28 bp, by AseI digestion, and the allele having the A385T mutation, sej, was separated into two fragments, 51 and 29 bp by AluI digestion. Although the faint residual bands, 98 bp by AseI digestion and 80 bp by AluI digestion, were sometimes observed depending on digestion conditions, we could clearly determine their zygosity by this experiment. As seen in PCR-RFLP of Family 1 in Fig. 4, TO, HN, and AN possessed the C357T mutation homozygously, whereas MO, SN, and TN were heterozygotes for this mutation. With regard to the A385T mutation, PCR-RFLP revealed that HN was a homozygote having this mutation in both alleles, and TO, TN, and AN were heterozygotes for this mutation. MO and SN did not have the A385T mutation in either allele.
All of the members, except for HN, in Family 1 were secretors with Le(a-b+) or Le(a-b-) phenotype. PCR-RFLP results revealed that all, except for HN, possessed at least one Se1 or Se2 allele. Only HN, a Le(a+b-) nonsecretor, in Family 1 was determined to be homozygous with sej/sej. The Sec2 genotype determined by PCR-RFLP is shown with initials of each individual in Fig. 4a. All the members of Family 2, except for HN who has a Le(a-b-) nonsecretor phenotype, were typed as Le(a+b-) nonsecretors. The results of PCR-RFLP on Family 2 as seen in the electrophoresed patterns clearly demonstrated that all the members of Family 2, including HN, were homozygous for the two mutations, C357T and A385T. Thus, all including YoN whose Sec2 gene was sequenced in the previous section, were determined to be homozygous for sej/sej. The PCR-RFLP methods that detected the two mutations revealed that the alleles were inherited according to genetic rules in all four families examined in this study. In conclusion, all individuals with the Le(a-b+) or Le(a-b-) secretor phenotypes had at least one of the Se1 or Se2 alleles, whereas all Le(a+b-) and Le(a-b-) nonsecretor individuals were homozygous for sej/sej.
As shown in Fig. 5, lysates of the
COS-1 cells transfected with the Se1 allele and those
transfected with the Se2 allele showed full
(1,2)fucosyltransferase activity, indicating that the Se1 and Se2 alleles encode a wild-type enzyme, while the sej-transfected COS-1 cell lysate showed quite low activity, i.e. about 2-3% of the activity of Se1- or Se2-transfected cell lysate. These results demonstrated that a
single amino acid substitution, Ile
to Phe, in the enzyme
directed by the sej allele causes almost complete inactivation
of the enzyme. Individuals with the sej/sej genotype are,
therefore, mutants who lack
(1,2)fucosyltransferase activity
directed by this gene.
Figure 5:
(1,2)Fucosyltransferase activity
directed by each of three alleles, Se1, Se2, and sej. From left to right, pCDM8, activity of lysate of
COS-1 cells mock-transfected with pCDM8 vector; pCDM8/Se1,
activity of Se1-transfected COS-1 cell lysate; pCDM8/Se2, activity of Se2-transfected COS-1 cell
lysate; pCDM8/sej, activity of sej-transfected COS-1
cell lysate. The activity in each lysate was normalized by transfection
efficiency which was determined by expression of co-transfected
luciferase expression vector. Data represent means ± S.D. of
three independent assays.
All of the results in this random sampling study are summarized in Table 2. The number of individuals with erythrocyte phenotype Le(a+b-), Le(a-b+) and Le(a-b-) were 31, 143, and 31, respectively among the 205 individuals randomly sampled. 31 Le(a-b-) individuals were divided into secretors (27 individuals) and nonsecretors (4 individuals) by the presence or absence of ABH antigens in saliva. The frequency of the four phenotypes, Le(a+b-), Le(a-b+), Le(a-b-) secretor and Le(a-b-) nonsecretor, were 15, 70, 13, and 2%, respectively. All 31 individuals with Le(a+b-) phenotype were determined to be homozygous for the sej/sej genotype by PCR-RFLP. All 143 individuals with Le(a-b+) phenotype were determined to have a genotype possessing at least one of the Se1 or Se2 alleles. 27 secretors among 31 Le(a-b-) individuals were also determined to have the Sec2 genotype possessing at least one of the Se1 or Se2 alleles, whereas all 4 non-secretors with Le(a-b-) phenotype were determined to be sej/sej homozygous.
The results showed that 31 individuals with the Le(a+b-) phenotype and 4 individuals with the Le(a-b-) nonsecretor phenotype among 205 randomly sampled individuals possessed the sej/sej genotype. This indicates a rather wide distribution of the sej allele having C357T and A385T mutations in the Sec2 gene.
With regard to the Le genotype determined by PCR-RFLP, 174 individuals with the Le(a+b-) or Le(a-b+) phenotype were determined to possess at least one Le allele that encodes an active Le enzyme, whereas 31 individuals with the Le(a-b-) phenotype were confirmed to be homozygous for the inactive mutant Le genes, i.e. le1 and le2. The results of Le genotyping on 205 individuals were completely consistent with previous results (3) in which we demonstrated that the Le alleles of the Japanese population could be classified into three alleles, Le, le1, and le2, without exception.
Even though an extensive molecular genetic analysis of the Sec2 gene was performed on the 45 nonsecretor individuals with the Le(a+b-) phenotype or the Le(a-b-) nonsecretor phenotype, including 10 individuals in the family study and 35 individuals in the random sampling study, we could not find any mutant allele other than sej.
The results described in this section demonstrated that combined genotyping of the Le and Sec2 genes can clearly distinguish the three Lewis blood group phenotypes, Le(a+b-), Le(a-b+), and Le(a-b-), with 100% accuracy. The results of this random sampling study (Table 2) indicate that the frequencies of occurrence of Le, le1, le2, Se1, Se2, and sej among 410 alleles examined in the Japanese population are 66, 27, 7, 15, 46, and 39%, respectively (Table 3). The frequencies of occurrence of Le, le1 and le2 were slightly different from the figures in the previous study (3) due to the increase in number of alleles examined in the present study.
In a previous study(3) , we demonstrated that the Le gene (Fuc-TIII gene) is solely responsible for the
expression of the Lewis blood group antigens, Le and
Le
, of erythrocytes. The Le alleles in a Japanese
sample were found to be divided in the three kinds, Le, le1, and le2. The latter two alleles possess
different point mutations and encode nonfunctional Lewis enzymes which
are inactivated by each single missense mutation in the catalytic
domain.
As proposed schematically in Fig. 1, the Le antigen on erythrocytes is a cooperative product of two
fucosyltransferases, an
(1,2)fucosyltransferase, which catalyses
the transfer of fucose to galactose with an
(1,2)-linkage and
synthesizes an H type-1 structure (O-type antigen), and the Lewis
enzyme with
(1,4)fucosyltransferase activity. Lewis antigens,
Le
and Le
, are known to be distributed not only
on erythrocytes but also in plasma, saliva, and other
secretions(9) . It has been reported that the Lewis antigens of
erythrocytes are not synthesized by the erythrocytes themselves but are
acquired from plasma by adsorption(8) . The major cells that
synthesize Lewis antigens are considered to be digestive organ
epithelial cells, where the two fucosyltransferases should be
localized. We recently established a monoclonal antibody that reacts
specifically with the human Lewis enzyme. An immunohistochemical study
using this antibody clearly demonstrated that the Lewis enzyme is
localized in digestive organ epithelial cells, but not in the cells of
myeloid lineage (results will be published elsewhere).
According to
the pathways proposed in Fig. 1which are presumed to function
in digestive organ epithelial cells, nonsecretor individuals who lack
Se enzyme activity would not synthesize the H type-1 structure that is
also a precursor structure of A, B, and Le antigens,
resulting in lack of secretion of ABH antigens into saliva or no
expression of Le
antigen in plasma or of erythrocytes. If
this is the case, Le(a+b-) and Le(a-b-)
nonsecretor individuals are likely to be mutants whose Se gene
is probably inactivated by point mutation.
It was proven by Kelly et al.(14) that the Sec2 gene is the the Se gene based on the finding of the G428A nonsense mutation in Sec2 gene in all six nonsecretors among 52 individuals
randomly sampled. This was confirmed in this study by discovery of the
A385T (Ile to Phe) missense mutation in the Sec2 gene of Japanese nonsecretors. This mutation is rather widely
distributed in the Japanese population, and this amino acid
substitution inactivated enzyme activity almost completely. It remains
to be elucidated whether the amino acid substitution is located at the
active sites of the enzyme, i.e. the acceptor- or the
donor-binding site. There is also the possibility that a single amino
acid substitution causes alteration of the tertiary structure of the
protein, resulting in marked susceptibility to proteolytic degradation
in the cells.
All Le(a+b-) and Le(a-b-)
nonsecretors examined were determined to be homozygous for sej/sej, whereas all Le(a-b+) and
Le(a-b-) secretors possessed active genes, Se1 or Se2, at least. Complete consistency between phenotypes and
genotypes in this extensive analysis of more than 200 individuals may
be enough to assert that the Sec2 gene is the Se gene. It was strongly suggested that the Se enzyme in the pathways
proposed in Fig. 1functions in digestive organ epithelial cells
in order to determine not only the secretion of ABH antigens into
saliva but also the expression of Le on erythrocytes.
The number of Lewis antigens on erythrocytes are far fewer than the ABH antigens. This is why we sometimes observe very weak hemagglutination by anti-Lewis antibodies, especially in certain conditions such as pregnancy and intestinal cancer. The typing of secretor status is also often ambiguous due to the low sensitivity of the assay method. The combination genotyping of Le and Se genes described in this study can be applied to ambiguous samples as a definitive typing method. Because of the higher frequency of occurrence of the Se2 allele compared with the Se1 allele, and because the C357T mutation found in the sej allele exists in the Se2 allele, we assume that the Se2 allele is the antecedent wild-type allele that gave rise to the Se1 and sej alleles afterward.
It was a quite interesting observation that all the Japanese nonsecretors examined had sej/sej homozygosity, making up more than 15% of the population. Similarly, a previous study by Kelly et al.(14) demonstrated that the Se enzyme-inactivating G428A mutation, which differs from the A385T mutation we found, is also rather widely distributed in another ethnic group, although they did not mention what ethnic group they analyzed. We never found the G428A mutation in Japanese individuals. Thus, these results suggest that Se gene inactivation must have occurred after the ethnic groups diverged.
An interesting question is why a sole inactivated allele spread in each ethnic group during a relatively short period. Kelly et al.(14) conjectured that the inactivated Se allele might confer some specific selective advantage upon the mutant individuals. Ethnic-specific alleles are not found in ABO genes, and these allelic mutations are conserved in all ethnic groups. The single inactivated sej allele in the Japanese population, even though it arose more recently than did the ABO alleles, already occupied a frequency of 39% among Se alleles. This situation might be similar to that of Le gene inactivation. This extensive study on 205 Japanese individuals (410 alleles) determined the frequencies of occurrence of two inactivated le alleles, le1 and le2, as 27 and 7%, respectively. Although the le2 allele has been found in Indonesian individuals(4) , the le1 allele having the G508A mutation, which occupies a much higher frequency than le2 in Japanese people, has not been found in other ethnic groups yet. This suggests that the le1 allele might be specific to the Japanese.
If the absence or the presence of Lewis antigens, now
proven to be the product of Le and Se genes, confers
some selective pressure upon the human species, it is conceivable that
antigen expression in digestive organs is biologically much more
important than the expression in erythrocytes. Recent investigation on Helicobacter pylori, a causative agent of gastric disorders (22, 23) , suggested that it resides in the stomach by
attaching to gastric epithelium via an H type-1 structure and a
Le antigen expressed thereon. Transgenic mice with the
human Le gene have been recently developed(23) , and
it was demonstrated that H. pylori obviously attached to
gastric epithelial cells in the transgenic mice but not in their normal
littermates. This implies that le/le individuals might have an
advantage in avoiding H. pylori infection.
Another possible biological significance of Lewis antigens in the digestive system concerns tumor-metastasizing capacity. The level of expression of sialylated Lewis antigens, sialyl Lewis a and sialyl Lewis x, on tumor cells is thought to correlate with metastasizing capacity(24, 25, 26) , since these antigens have been shown to be the ligands to E-selectins(27) . Some statistical results (26) have indicated that the more sialylated Lewis antigens tumor cells have, the more malignant the cells are. If the Le enzyme and the Se enzyme are also involved in the pathway of sialylated Lewis antigen synthesis in intestinal cancer cells, the genotypes of the Le and Se genes of cancer patients would affect their prognosis. Further study might analyze statistical correlations of the Le and Se genotypes with H. pylori infection and with the metastasizing capacity of cancer cells.