Hum Genet (2000) 106 : 8085

Digital Object Identifier (DOI) 10.1007/s004399900212

O R I G I N A L I N V E S T I G AT I O N

Yoshiro Koda Mikiko Soejima Philip H. Johnson

Elizabeth Smart Hiroshi Kimura

An Alu-mediated large deletion of the FUT2 gene

in individuals with the ABO-Bombay phenotype
Received: 8 October 1999 / Accepted: 13 November 1999 / Published online: 18 December 1999
Springer-Verlag 1999

Abstract Recently, we have found an allelic deletion of

the secretor (1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO
system. The FUT2 gene consists of two exons separated
by an intron that spans approximately 7 kb. The first exon
is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5 breakpoint of the deletion has
previously been mapped to the single intron of FUT2, we
have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3 untranslated region of FUT2 to the 3 breakpoint sequence has been amplified from a control individual. DNA sequence analysis
of this region indicates that the 5 breakpoint is within a
free left Alu monomer (FLAM-C) sequence that lies 1.3
kb downstream of exon 1, and that the 3 breakpoint is
within a complete Alu element (AluSx) that is positioned
1.5 kb downstream of exon 2. The size of the deletion is
estimated to be about 10 kb. There is a 25-bp sequence
identity between the reference DNA sequences surrounding the 5 and 3 breakpoints. This demonstrates that an
Alu-mediated large gene deletion generated by unequal
crossover is responsible for secretor (1,2)fucosyltransferase deficiency in Indian Bombay individuals.

Y. Koda M. Soejima H. Kimura ()

Department of Forensic Medicine and Human Genetics,
Kurume University School of Medicine, Kurume,
Fukuoka 8300011, Japan
e-mail: hkimura@med.kurume-u.ac.jp,
Tel.: +81 942 31 7554, Fax: +81 942 31 7700
P. H. Johnson
MRC Blood Group Unit, 4 Stephenson Way,
London NW1 2HE, UK
P. H. Johnson
Molecular Genetics, Department of Cardiothoracic Surgery,
National Heart and Lung Institute, Harefield,
Middlesex UB9 6JH, UK
E. Smart
Immunohaematology, Natal Blood Transfusion Service,
Private Bag X9044, Pinetown, 3600, Republic of South Africa

Introduction
Tissue expression of the ABH antigens is regulated by
several glycosyltransferases (Watkins 1995). Alpha(1,2)fucosyltransferase, which forms the H antigen (a precursor
of the A and B antigens) is essential for the tissue expression of ABH antigens. Two distinct (1,2)fucosyltransferases are known to be present in human tissues. One is
the H gene (FUT1)-encoded (1,2)fucosyltransferase (H
enzyme), which regulates the expression of the H antigen
on erythrocyte membranes, and the other is the Secretor
gene (FUT2)-encoded (1,2) fucosyltransferase (Se enzyme), which regulates the expression of the H antigen in
the secretory fluids and digestive mucosa (Oriol et al. 1986;
Clausen and Hakomori 1989; Liu et al. 1998a, 1999a). The
frequency of H enzyme deficiency (Bombay and paraBombay phenotypes) is very low (e.g., 1 in 13,000 Indians and 1 in 312,081 Germans; Bhatia and Sanghvi 1962;
Wagner and Flegel 1997). In contrast, Se enzyme deficiency (nonsecretor phenotype) is about 20% in African,
European, and Asian populations (Koda et al. 1996; Liu et
al. 1998b). Individuals with the Bombay phenotype fail to
express ABH antigens either on red cells or in secretions,
because they lack both H and Se enzyme activities and are
known to have anti-H, anti-A, and anti-B antibodies in
their serum (Daniels 1995; Watkins 1995).
FUT1 and FUT2 have previously been isolated (Larsen
et al. 1990; Kelly et al. 1995; Rouquier et al. 1995). In addition, we have determined the gene structure of FUT2
(Koda et al. 1997a). The results indicate that FUT2 consists of two exons that are separated by a 7-kb intron sequence. The first exon is a noncoding exon, whereas exon
2 contains the complete coding region. The sequencing of
the intervening intron and of the 3 untranslated region
have demonstrated the presence of numerous Alu elements (Koda et al. 1997a).
The molecular mechanism for the deficiency of H and
Se enzymes has been analyzed in many populations
(Kelly et al. 1994, 1995; Koda et al. 1996; Yu et al. 1996;
Kaneko et al. 1997; Wang et al. 1997; Wagner and Flegel

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Table 1 Sequences and positions of PCR primers (numbering as in Koda et al. 1997a and Fig. 1)
Primers

Sequences

Positions (bp)

Bombay-U-first
Bombay-U-nest
3 UTR
Bombay-L

5-CCT CCC CAG GGT GGA AGT GAT AAT G-3

5-GCG GAG AGC TGG GTT ATT TCA CGG GAA CAG-3
5-CCC CAC AGC AGC CTT CCC TCT CAG A-3
5-GTG TCT CGG GCA CTC GTC TTT CAG C-3

593~617, within intron

773~802, within intron
2659~2673, within exon 2
1227~1251, 3 of the 3 breakpoint

1997; Fernandez-Mateos et al. 1998; Liu et al. 1998b,

1999b). These results have demonstrated that the frequency of H enzyme-deficient alleles is very low, and that
there is no prevalent null allele, whereas the frequency of
Se enzyme-deficient alleles is about 50% in all populations tested, and there are prevalent population-specific
null alleles.
In our previous study, we have found that a T-to-G
base substitution at position of 725 bp of FUT1 (T725G,
numbering as in Larsen et al. 1990), which results in an
amino acid change (leucine to arginine) at codon 242, and
a complete deletion of FUT2 (sedel) are responsible for the
classical Bombay phenotype (Koda et al. 1997b). In this
study, we have isolated the junction region of the deletion
of FUT2 by cassette-mediated polymerase chain reaction
(PCR). The results from DNA sequencing suggest that the
deletion is generated by Alu-Alu recombination. We have
also developed a PCR method to identify the sedel allele.

Materials and methods

DNA preparation
Genomic DNA from three unrelated Indian individuals with the
Bombay phenotype and from control individuals was extracted
from peripheral leukocytes by the organic solvent method (Koda et
al. 1997b).
PCR amplification of the junction region of the sedel allele
A Marathon cDNA amplification kit (Clontech) was used for the
isolation of the junction region of the deletion. Genomic DNA (5
g) from a Bombay individual was digested with one of several
endonucleases (50 U; DraI, PvuII, RsaI, and EcoRV) and ligated
to the Marathon cDNA adaptor. Since AP1 and AP2 sequences are
present within the adaptor, the junction region of the deletion was
isolated by PCR amplification with AP1 and AP2 primers (Clontech) as upstream primers. The nucleotide sequences and positions
of primers are indicated in Table 1, Fig. 1A. The Bombay-U-first
primer and the AP1 primer were used for the first PCR and then
nested PCR was performed by using the Bombay-U-nest primer
and the AP2 primer. The temperature profile of all PCRs for the
amplification of the junction region of sedel was 94C for 1 min followed by 25 cycles of denaturing at 98C for 10 s and annealing/extension at 68C for 4 min. PCR amplification was performed
by using 2.5 U LA Taq polymerase (Takara) in 25 l LA Taq
buffer containing 5 pmol each primer, 2.5 mM MgCl2, and 400 M
dNTPs.
PCR amplification of a region encompassing the 3 breakpoint
A DNA fragment encompassing the 3 breakpoint of the deletion
was amplified from the genomic DNA of a control individual by
using the 3 UTR primer and the Bombay-L primer. The tempera-

ture profile was 94C for 1 min, followed by 30 cycles of denaturing at 98C for 10 s, annealing at 60C for 30 s, and extension at
72C for 5 min. PCR amplification was performed by using 2.5 U
LA Taq polymerase in 25 l LA Taq buffer containing 5 pmol
each primer, 2.5 mM MgCl2, and 400 M dNTPs.
Detection of the sedel allele by PCR
PCR to amplify the sedel allele was performed in 25 l Ex Taq
buffer containing 5 pmol Bombay-U-nest and Bombay-L primers,
1 U Ex Taq DNA polymerase (Takara), 200 M dNTPs, and genomic DNA from Bombay or control individuals. The temperature
profile was 94C for 1 min, followed by 35 cycles of denaturing at
98C for 10 s, annealing at 60C for 30 s, and extension at 72C
for 2 min.
DNA sequencing
PCR products were purified by centrifugation in Suprec-02 tubes
(Takara) and were then directly sequenced in both directions by
using each PCR primer or several internal sequencing primers (not
shown) with a Bigdye Terminator Cycle Sequencing Reaction Kit
and an ABI PRISM 310 genetic analyzer (Perkin Elmer Japan ABI).

Results
Identification of the junction region of the sedel allele
Previous mapping results suggested that the 5 end of the
gene deletion was located in a 7-kb region within the single intron of the FUT2 gene (Koda et al. 1997b). To determine the position of the 5 breakpoint in more detail,
several parts of the intron were amplified by using genomic DNA from an Indian Bombay individual as a template (data not shown). These results suggested that the 5
breakpoint was located between 816 bp and 1434 bp into
the FUT2 intron. Then, cassette-mediated PCR was performed to clone the junction region of the deletion. The
positions of primers used for cassette-mediated PCR are
indicated in Fig. 1A. The longest PCR product (2 kb) was
obtained from a DraI library. DNA sequence analysis indicated that a 571 bp sequence at the 5 end of the product
was identical to the previously obtained intron sequence
of FUT2 (from positions +773 to +1340), except for the
length of the poly A tail on the first Alu element in the intron (27 bp in the Bombay individual as compared with 24
bp in a reference sequence). The remaining 1399 bp at the
3 end of the DraI product has not been identified previously (Fig. 2). Therefore, the 5 breakpoint of the deletion
is positioned 1.3 kb downstream of the 3 end of exon 1.
The 5 breakpoint is within an Alu element, suggesting

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Fig. 1 A The physical map of the deleted region (Reference allele)

and the junction region of the sedel (Deletion allele). Blank boxes
indicate exons of the FUT2 gene. Shaded and black boxes indicate
Alu elements present in this region. The positions of primers are
indicated by arrows. B The alignment of the junction region (Junction) with the free left Alu monomer (FLAM-C) sequence surrounding the 5 breakpoint (5 Alu) and the Alu left arm (AluSx) sequence surrounding the 3 breakpoint (3 Alu). Shaded boxes indicate the 26-bp Alu core sequence (CCT GTA ATC CCA GCA
CTT TGG GAG GC), which is considered to be a recombination
hotspot (Rudiger et al. 1995). Identical nucleotides in sequence are
indicated by asterisks

that Alu-Alu recombination may be responsible for this

large gene deletion in the FUT2 gene of classical Bombay
individuals.
Sequencing of a region between the 3 untranslated region
of FUT2 and the 3 breakpoint in a control individual
Subsequently, we attempted to amplify the region between
the 3 untranslated region of FUT2 and the position of the
3 breakpoint in the sedel allele by using genomic DNA from
a control individual as a template. The resulting 3-kb PCR
product was confirmed by DNA sequence analysis as the

3 flanking region of the FUT2 gene. Furthermore, the position of the 3 breakpoint of the deletion was identified
precisely 1.5 kb downstream of the 3 end of exon 2 (the
last exon) of FUT2. In our previous study (Koda et al.
1997a) and in the present work, we determined the entire
13.5-kb genomic DNA sequence from a promoter region
to the 3 flanking region of the FUT2 gene. From these results, the size of the deletion in the FUT2 gene was estimated to be about 10 kb (Fig. 1A). Alu elements in the
13.5-kb DNA sequence of FUT2 were identified by using
the Repeat Masker program from the Washington University Human Genome Center (http://ftp.genome.washington.edu/cig-bin/). These results indicated that both the 5
and 3 breakpoints were located within Alu elements. An
alignment of reference DNA sequences flanking the 5
and 3 breakpoints and the sequence of the junction region
revealed a homologous sequence of 25 bp, indicating that
the deletion occurred by Alu-Alu homologous recombination (Fig. 1A, B). The 26-bp Alu core sequence (CCT
GTA ATC CCA GCA CTT TGG GAG GC), considered
to be a recombination hotspot (Rudiger et al. 1995), was
present in the junction region. We found 23 partial and
complete Alu elements (total 5.8 kb) in the 13.5-kb DNA
sequence from the promoter region to the 3 flanking re-

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Fig. 2 DNA sequence of a cassette-mediated PCR product containing the junction of the deletion of the FUT2 gene. The positions of the PCR primers (Bombay-U-nest and Bombay-L) are underlined. Shaded boxes indicate Alu elements. The presumed recombination junction point is indicated by a slash

primers encompassing the junction of the deletion. The

position of the PCR primers is shown in Fig. 1A and Fig.
2. As shown in Fig. 3, a 1.8-kb DNA fragment was amplified from the genomic DNA of three Indian Bombay individuals homozygous for the sedel allele. No product was
amplified from the genomic DNA of control individuals.
Direct DNA sequencing of the 1.8-kb PCR products from
three Bombay individuals indicated specific amplification
across the junction of the deletion.

Discussion

Fig. 3 PCR amplification of the junction region of the sedel. PCR

products underwent electrophoresis in a 1.2% agarose gel and
were stained by ethidium bromide. The size of the PCR product is
indicated right. Lane 1 Control individual, lanes 24 Indian Bombay individuals. StyI-digested phage (M) was used as a molecular size marker

gion of the FUT2 gene, which suggests that Alu-mediated

recombination is an important causative mechanism for
large deletions in the FUT2 gene.
Identification of the sedel allele by PCR
For further confirmation of the gene deletion, we amplified the sedel allele by a conventional PCR method with

We report here an Alu-mediated large genomic deletion of

the FUT2 gene in classical Bombay individuals. The 5
breakpoint of the deletion is positioned 1.3 kb downstream of the 3 end of exon 1, and the 3 breakpoint of
the deletion is located 1.5 kb downstream of the 3 end of
exon 2 of the FUT2 gene. The size of the deletion FUT2
is calculated to be about 10 kb. The 1.1-kb complete coding region located within exon 2 is oblated by this large
rearrangement. The human genome contains about 1106
copies of Alu repeats distributed throughout the genome
with an average spacing 4 kb (Hwu et al. 1986). The computer search analysis has revealed that 43% (5.8 kb) of the
13.5-kb region from a promoter region to the 3 flanking
region of FUT2 consists of 23 partial and complete Aluelements (Fig. 1A). The density of Alu elements in this region is high. These sequences are known to be hotspots for
large rearrangements, such as deletions and duplications, in
genomic DNA. Accordingly, the FUT2 locus may be particularly susceptible to Alu-mediated DNA rearrangements.
Generally, large genomic deletions may remain undetected by conventional PCR-based methods in heterozy-

84

gous cases, because of amplification of the undeleted allele. As an example, a recent study of BRCA1 has demonstrated that initial screening by conventional PCR-based
methods, such as single-strand conformation polymorphism analysis, the protein truncation test, and direct sequencing, have failed to detect two different Alu-mediated
deletions that are present with a frequency of 36% in
Dutch kindreds (Petrij-Bosch et al. 1997). A 3.5-kb Alumediated deletion of MLH1 (Nystrm-Lahti et al. 1995)
and a 1-kb Alu-mediated deletion of BRCA1 exon 17
(Puget et al. 1997) have also been found to be a frequent
type of mutation in cancer susceptibility genes. In addition, we have found a fusion gene consisting of a FUT2
pseudogene and FUT2 as a null allele, generated by unequal crossover in the Japanese population and present at
a relatively high frequency (more than 5%; Koda et al.
1996; Liu et al. 1999b). Since the frequency of FUT2 deficiency (nonsecretor phenotype) is particularly high (more
than 20% in many populations), it is possible that several
different gene recombination events may have occurred at
this locus. In addition, gene duplications may also be generated as a reciprocal product of gene deletion by an unequal crossover event. Therefore, PCR amplification of
the coding region followed by conventional mutational
analysis is not sufficient to detect large deletions of the
FUT2 gene responsible for the nonsecretor phenotype.
In this study, we have developed a method for identifying the sedel allele by using PCR. This method can detect
sedel in individuals who have been demonstrated to be homozygous for the sedel by Southern blot analysis. Previous
studies have indicated that the T725G mutation of FUT1
is consistently linked with the sedel of FUT2 in individuals
with the classical Bombay phenotype (Koda et al. 1997b;
Fernandez-Mateos et al. 1998). Therefore, this PCR
method may be useful for detecting the sedel allele, other
types of large deletion in FUT2, and linkage between the
T725G mutation of FUT1 and the sedel allele of FUT2.
Acknowledgements This work was supported in part by Grantin-Aids for Scientific Research from the Ministry of Education,
Science, Sports, and Culture of Japan, and a grant from the Uehara
Memorial Foundation. Nucleotide sequence data reported in this
paper have been deposited in the DNA Data Bank of Japan
(http://www.DDBJ.nig.ac.jp/) with accession nos. AB032485 (the
junction region of the sedel allele of FUT2) and AB032486 (the 3
flanking region of FUT2).

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