Blackwell Science, LtdOxford, UKTRFTransfusion0041-11322004 American Association of Blood BanksMay 2004445707715Original ArticleEVOLUTION OF HUMAN O ALLELESROUBINET ET AL.

IMMUNOHEMATOLOGY
Evolution of the O alleles of the human ABO blood group geneFrancis Roubinet, Stéphanie Despiau, Francesc Calafell, Fen Jin, Jaume Bertanpetit, Naruya Saitou,
and Antoine Blancher

BACKGROUND: To date, at least 40 different alleles O
have been characterized on the basis of exon 6 and exon
7 sequences but not always for intron 6.
STUDY DESIGN AND METHODS: Among 415
individuals, from four continents (Africa, Europe,
South America, and Asia), studied for exon 6 andexon 7 sequences, we selected 46 individuals (ofrespective phenotypes O [39], AB [3], B [3], or A [1])for sequencing 1800-bp amplicons spanning exon 6,
intron 6, and exon 7. The amplicons were characterized
either by direct sequencing or after cloning whenrequired.
RESULTS: We defined 14 new intron 6 O allele
sequences, including four recombinant alleles. Basedon sequence comparison, a phylogenetic network
was constructed. It confirmed recombinant allele
origins and that most O alleles are derived by point
mutations from the two worldwide distributed alleles
O01 and O02.
CONCLUSION: Allele O phylogenetic analysis suggeststhat the most frequent silencing mutation (deletion of a G
in exon 6) appeared once in human evolution in theancient O02 allele lineage and that allele O01 resulted
from an interallele exchange between O02 and A101.
Assuming constancy of evolutionary rate, diversificationof the representative alleles of the three human ABO
lineages (A101, B101, and O02) was estimated at 4.5 to6 million years ago.

T

he ABO system was discovered by Landsteiner in
1901.1 It is the most important blood group system in transfusion and transplantation practices. It was also the first human genetic system
to be applied to anthropologic studies. The A and B allelesof the ABO gene code for glycosyltransferases that either
add an N-acetylgalactosamine or a galactose to various
glycoconjugates. These products result in A or B bloodgroup-specific antigens. The O allele corresponds to asilent (null) allele of the ABO gene. The cDNAs of the threemajor alleles of the human blood group ABO system werefirst described by Yamamoto and coworkers.2,3 The human
ABO locus spanning 18 kb, lies on chromosome 9q34 andencompasses seven exons giving a 1062-bp open reading
frame. Exons 1 through 5 encode the amino terminal part,a transmembrane region, and 9 percent of the catalytic
domain. Exons 6 and 7 encode 77 percent of the protein
and 91 percent of the catalytic active part.4 Because of

From the Laboratory of Molecular Immunology, Paul Sabatier
University, Rangueil Hospital, 31403 Toulouse Cedex 4, France;
the Laboratory of Immunohematology, French Establishment ofTransfusion of the Pyrenes-Mediterranean, Toulouse Site, Avenueof Great Britain, BP 3210, 31027 Toulouse Cedex, France; Unit of
Evolutionary Biology, Faculty of Health Life Sciences, Pompeu
Fabra University, Doctor Aiguader, 80, 08003 Barcelona,
Catalonia, Spain; the Institute of Genetics and Developmental
Biology, Academia Sinica, Beijing, China; and the Division ofPopulation Genetics, National Institute of Genetics, Mishima,
411-8540, Japan.
Address reprint requests to: Antoine Blancher, MD, PhD,
Laboratoire d’Immunogénétique Moléculaire, Université Paul
Sabatier, Hôpital Rangueil, Faculte de Medicine Bâtiment A2, 133
Route de Norbonne, 31062 Toulouse Cedex 4, France; e-mail:blancher@easynet.fr.
This work was supported by funds from the Agence
Française du Sang (Contract 97002551231) from theEtablissement Français du Sang and from the Ministère desAffaires Etrangères, France (Contract EA 3034).
Received for publication October 27, 2003; revision received
December 20, 2003, and accepted January 8, 2004.
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that, ABO gene polymorphism has been studied, almost
exclusively, in exons 6 and 7.
Since this pioneering period, many variants of theclassical A and B alleles have been characterized.5,6 Moreover, numerous inactive (O) alleles have been reported.5,7,8
The high diversity of inactive alleles in this gene gives aprecious opportunity to study convergent loss of function
and the role of selection in the maintenance of such diversity in populations.
The most frequent human O alleles, O01 and O02(http://www.bioc.aecom.yu.edu/bgmut/abo.htm), differ
in exons 6 and 7 by nine nucleotide substitutions2,9 but
share one nucleotide deletion of a G at position 261 inexon 6 of the A101 sequence (D261).5 This deletioninduces a frameshift and creates a premature stop codon(nucleotides 352-354), resulting in a truncated (117amino acids) protein deprived of any glycosyltransferase
activity.2 Numerous variants of O01 and O02 alleles havebeen described. They differ from O01 or O02 by a few
point mutations.5,7,8,10-14 A rare silent allele, initially called“another O allele”15 or “O2”16 now referred to as O03, wasobserved only in Europeans and at low frequencies in
sub-Saharan Africans.10-12,17-21 Allele O03 does not contain
the G261 deletion but carries a nonsynonymous inactivating mutation in exon 7.15,22 Another rare null allele,23
initially called O3, and also referred to as O08,5 differsfrom O01 and O02 by the absence of the D261 mutation.
Allele O08 is basically an A2 allele in exons 6 and 7 but
displays an inactivating mutation in exon 7, which consists of an insertion of a G in a stretch of G’s (between nt
798 and 804).23 More recently, two other inactivating
mutations were identified in very infrequent silent alleles:
allele O4 has a G insertion at nt 87 to 88 in exon 2 of aA101 allele resulting in a premature stop codon (codon
56), and allele O5 displays a non-sense mutation in exon
6 (C322T).7,24
We previously studied the polymorphism of the O
allele in five human populations of three continents,
Europe, Africa, and South America.12 Nevertheless, theABO blood group gene polymorphism was characterized by direct sequencing of exons 6 and 7 only. In
this study, we extend the analysis of O alleles encountered in the five populations of our previous study bysequencing fragments amplified from the 3¢ end of
intron 5 to the end of exon 7 (including intron 6),
which, as discussed below, is crucial to properly
resolve the phylogeny reconstruction of the allelic lineages of this locus. Two Chinese populations of O
phenotypes were added in the study. We also analyzed
the phylogenetic relationship between human O alleles
and the two main functional alleles A101 and B101 tounderstand the molecular mechanisms that have generated each O allele. This analysis gives a more complete understanding of the diversity and origins of O
alleles.
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MATERIALS AND METHODS
Samples
We used the same human samples as those described in
our previous study of exons 6 and 7,12 that is, one Akan
individual of B phenotype and 317 unrelated individualsof O phenotype from five human populations, comprising
Akans from Ivory Coast, Moroccan Berbers, Basques from
Spain and France, and Cayapas and Aymaras from Bolivia.
We also studied 6 Caucasian blood donors from the Toulouse area (southwest France) and one Akan individual tocharacterize sequences of 14 common alleles (4 A alleles,
5 B alleles, 2 O01, 2 O02, and 1 O03). Furthermore, individuals of O phenotype from two Han populations in
China (47 individuals at Putien in Fujien Province and 43
individuals at Fujiou in Guandong Province, both Han
Chinese) were newly examined in this study.
Among these 415 individuals, studied for exon 6 andexon 7 sequences, we selected 46 individuals (of respective phenotypes O [39], AB [3], B [3], or A [1]) for sequencing 1800-bp amplicons spanning exon 6, intron 6, andexon 7. Among individuals of O phenotype for which DNA
samples were available, we selected individuals having O
alleles of each type.

Characterization of ABO alleles
Genomic DNA extraction and PCR amplification of aregion encompassing the end of intron 5 to the end ofexon 7 were performed as indicated in Roubinet et al.12
Primer locations are indicated in Fig. 1. Primers int.5-dirand 3¢UTR-rev were used to amplify the genomic fragment
encompassing the end of intron 5, exon 6, intron 6, andexon 7, in 30 cycles of PCR amplification with a 2.5-minuteelongation step. PCR products were cloned into thepCR2.1 Topo plasmid vector (Topo TA cloning Kit, Invitrogen, Leek, the Netherlands).12 Cloned products weresequenced using the fluorescent dye terminator method(Amplitaq FS, PE Applied Biosystems, Foster City, CA) as
previously described.12
The sequences obtained by cloning amplicons of agiven individual were aligned and compared to characterize the two alleles of each individual. As direct sequenceswere compatible with those deduced from combination ofalleles defined by cloning, 17 individuals were studied
only by direct sequencing of amplicons from exon 6 toexon 7 (including intron 6). A phylogenetic network was
manually constructed as in Saitou and Yamamoto.25

Nomenclature of alleles
Alleles were named following the unofficial nomenclatureof Yamamoto (http://www.bioc.aecom.yu.edu/bgmut/
abo.htm) followed between parentheses, when useful, by
the original names given by the authors who described theEVOLUTION OF HUMAN O ALLELES

Fig. 1. Schematic representation of the genomic organization of
the human ABO gene including intron 5, exon 6, intron 6, andexon 7 with primer positions. Primer sequences: int.5-dir,
TGATTTGCCCGGTTGGAGTCGC; int.6.1055-dir, GCTCACTGACCAGAGATAGC; int.6.1255-rev, AGTATGTGTCTGCGGTTGC;
Ex6.306-dir, CGACATCCTCAACGAGCAGTTC; Ex7.663-rev,
GGAGTCAGGATCTCCACG; Ex7.460-rev, CGGTGAAGACATAGTAGTGGACACG; and 3¢¢UTR-rev, CCAGAGCCCCTGGCAGC
CGCTC. 168, 135, 1052, and 688 correspond to numbers ofbases of the analyzed regions (respectively end of intron 5, exon
6, intron 6, and exon 7).

TABLE 1. Distribution of O genotypes among the two
Chinese populations
Population
Putien, Fujien
Fujiou, Guangdong

Total
47
43

O01/O01
13
11

Genotype
O01/O02
29
22

O02/O025
10allele for the first time. When alleles are not nomenclatured by Yamamoto we gave only the names given by theoriginal authors.

RESULTS
Nucleotide sequences
Direct sequences obtained from all 90 Chinese individuals
studied allocate them all in just three genotypes: O01/O01,
O02/O02, or O01/O02 (Table 1). Genotype frequencies
within O individuals were in Hardy-Weinberg equilibrium.
Relative allele frequencies were 0.59 O01 and 0.41 O02 in
Putien and 0.51 O01 and 0.49 O02 in Fujiou. All alleles
encountered in the eight populations, but two(021(O1(C467T) and O1v(A681G&C1054T)) were characterized bydirect sequencing and cloning (for at least one allele) fromexon 6 to exon 7, including intron 6 (Table 2). Individuals
studied by cloning are given without parentheses in Table
2. In total we characterized 24 ABO alleles from exon 6 toexon 7. Of these alleles, 15 (14 O alleles and 1 variant of
B101 allele) had never been characterized at the level ofintron 6. These sequences (comprising the total of exons
6 and 7 and intron 6) are shown aligned in Fig. 2 and allele
frequencies in the different population samples are shownin Table 3.
All four sequences of A1 alleles studied were identicalto the human genome sequence AC000397 (GenBank

accession number), which is consistent with an A101
allele.7 As for B alleles, five sequences were identical toallele B101 from exon 6 to exon 7. The sixth individual hada B allele (B101-var1) displaying one nucleotide difference
in intron 6, when compared to allele B101 (see Fig. 2).
By reference to our previous study,12 polymorphism in
intron 6 sequences splits three previously determined
alleles into two subtypes: O30 (Ovar.tlse05) is split into
Ovar.tlse51 and Ovar.tlse52, O24 (O1v-B) into O1v-B.tlse13
and O1v-B.tlse14, and O13 (Ov7) into Ov7.1 and Ov7.2. Moreover, the analysis of intron 6 led us to conclude that allele
O06 described in the previous study12 was in fact allele
O02. This misidentification of O06 alleles was due to misinterpretation of direct sequencing results of exon 6 amplicons at the time of our previous study. Indeed, taking
into account only e6 and e7, allele O06 differs from O02
only at position 297 of e6.
An 8-bp motif is tandemly repeated three times inintron 6, with one additional repeat in three human alleles (O1v-B.tlse14, Ovar.tlse52, and Ov7.2; see Fig. 2); thismay be considered to be an emergent microsatellite. The
mutation had a single origin in allele Ov7.2 and recombination took it into the other alleles. The same motif has
been found repeated three to five times in chimpanzee26
and three times in gorilla (F. Roubinet, unpublisheddata).

Recombination events
Some alleles carry sequences that have clearly originatedfrom recombination; these are stretches of identity or near
identity with one allele followed by a stretch of identity
or near identity with a different allele. These segments
usually contain three or more informative polymorphic
positions, making mutation as the origin of the allele
extremely unlikely. In this study four new recombinant
alleles were characterized, and we encountered alleles
O03 and O05 already characterized as recombinants.27,28
Five of these alleles (O1v-B.tlse13, O1v-B.tlse14,
Ovar.tlse51, O03, and O05) and their two hypotheticalparental alleles are depicted in Fig. 3.
Allele O1v-B.tlse13 can be derived either by two mutations from B101 or by a single recombination event
between O02 and B101. If it originated from two mutations from allele B101, one would be the deletion of a G at
position 261 in exon 6. As discussed below, it is highly
probable that this deletion occurred only once in allele
A101 (giving rise to allele O01). The hypothesis of a recombination involving alleles O02 and B101 between positions
i42 and i88 thus seems more probable (for numbering of
nucleotide positions see the legend to Fig. 2). This recombinant seems similar to O1v-7 allele, previously reported ina German family by Bugert et al.29
The O1v-B.tlse14 allele could have derived from point
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TABLE 2. Determination of genotypes
Individual genotypes
Identification of allele 1
A101
A101
O01
O01
O02
O02
O02
O02
O03
O03
O09
O09
O09
O11
O11
O12
O1v-B.tlse13
O1v-B.tlse13
O1v-B.tlse13
O1v-B.tlse13
O1v-B.tlse14
Ovar.tlse20
Ovar.tlse20
Ovar.tlse20
O26
O27
O28
O29
O33
O34
Ovar.tlse51
Ovar.tlse52

A
A
1
1
2
2
2
2
3
3
9
9
9
11
11
12
13
13
13
13
14
20
20
20
26
27
28
29
33
34
51
52

Identification of allele 2
B101
B
B101-var1
Bv
B101
B
O01
1
O02
2
A101
A
B101
B
O01
1
O01
1
O02
2
O01
1
O02
2
O31
31
O32
32
O11
11
O01
1
O02
2
7
Ov7.1
O28
28
O01
1
O01
1
7
Ov7.1
8
Ov7.2
O02
2
O02
2
B101
1
7
Ov7.1
O01
1
O02
2
O01
1
O01
1
O01
1

Individuals numbers and origin*
2, Toulouse
1, Toulouse
1, Toulouse(1, Bolivian and 2 Chinese)(2, Chinese)
1, Toulouse
1, African(1, Bolivian)
1, Toulouse cloned (1, Berbers, 2, Basques)(2, Berbers)
1, African
1, Basque1, African
1, Bolivian(1, Bolivian)
3, Basques
1, African
1, African
2, Africans(1, Basque)1, African
1, African (1, African)
1, African (1, African)(2, Africans)
1, Basque1, African
1, African
1, African
1, Bolivian
1, Basque1, Berber
1, African* Outside parentheses are listed the individuals studied by direct sequencing and cloning for E6-i6-E7 region. Between parentheses are listed
the individuals studied only by direct sequencing.

tion of an 8-bp repeat in intron 6) or from Ov7.2 (10 mutations). Nevertheless, a more parsimonious hypothesis is arecombination between Ov7.2 (derived from O02) and B101
(recombination site between positions i1014 and e525). It
should be noted that the O1v-B.tlse13 and O1v-B.tlse14
alleles are identical in exon 6 and 7 to the allele previously
named O1v-B, whose intron 6 sequence had not beensequenced before this study.12,30
The third new recombinant allele characterized here
is Ovar.tlse51. It could have derived from Ov7.1 by six point
mutations but it more probably derived from a single
recombination between O01 (5¢) and Ov7.1 (3¢) at a site
between positions i236 and i445. Interestingly, Ovar.tlse51
allele is observed in African populations, where the twoparental alleles Ov7.1 and O01 are frequent.
Another candidate having originated by recombination is allele Ovar.tlse20. If one excludes intron 6, it is identical to allele O05, which is a recombinant allele between
alleles O02 and O01 or A101.11 Nonetheless, allele
Ovar.tlse20 differs clearly from allele O05 in intron 6 (ninesubstitutions).11 Because allele Ovar.tlse20 carries threemutations not seen in other alleles, as well as three apparent reversions of O02 mutations and one singleton, it
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could correspond to a recombinant allele between O01
and a subtype of O02 not yet sampled or to an ancient
allele that has had a long time to accumulate mutations.
Allele O03,14,27 previously described as a complex
recombinant, has also been found. We confirmed that aprobable recombination site between B101 and A101 lies
between e527 and e656.

Phylogenetic network of human alleles
We constructed a phylogenetic network (Fig. 4) for 26
human ABO blood group allele sequences, 23 being O alleles, to examine the internal relationship based on variant sequence information shown in Fig. 2. The position of
the root of this phylogenetic network was estimated bycomparing chimpanzee and gorilla sequences.26 As analyzed above, six sequences (Ovar.tlse51, O1v-B.tlse14, O1vB.tlse13, Ovar.tlse20, O05, and O03) are compatible with arecombinant origin seen as complex reticulations, a clear
footprint of recombination. The two (Ovar.tlse51 and O1vB.tlse14) have not been incorporated in the network
because of the complexity they generate in the graph. Taking into account only sequences from exon 6 to exon 7,

EVOLUTION OF HUMAN O ALLELES

Fig. 2. Multiple alignments of human ABO blood group gene O allele sequences found in this study, with A101 and B101 major
functional alleles. The sequences were aligned through Clustal W and are given by reference to A101 allele. Sequence AF448499 is
identical to the human genome sequence AC000397. Nevertheless, the latter reference does not mention the ABO type of the individual analyzed. Therefore, no A101 allele sequence from exons 6 to 7 (including the intervening intron 6) was available in the databanks.
For that reason, we have deposited our sequence under Accession Number AF448499. Points signify identity with the reference anddashes indicate missing bases. The numbering of nucleotide positions in exons 6 and 7 is given by reference to cDNA numbering (theA of the start codon being 1). As for intron 6 the numbering is given by reference to intron 6 of allele A101 (position 1 being the firstbase of the intron). The insertion of 8 bp in certain alleles is not taken into account for the numbering. In the text, nucleotide positions
are given by reference to that used in the figure: if the position belongs to an exon, numbers are preceded by the letter “e,” and if theposition belongs to intron 6, the numbers are preceded the letter “i”. *The sequence was deduced from the publication of Ogasawara
et al.11

TABLE 3. O allele frequencies in different human populations*
Population
sample
1
2
3
5
7
8
9
11
12
13
14
20
21
26
27
28
29
31
32
33
34
51
52
53

Identificationof alleles
O01
O02
O03
O05
Ov7.1
Ov7.2
O09
O11
O12
O1v-B.tlse13
O1v-B.tlse14
Ovar.tlse20
O21
O26
O27
O28
O29
O31
O32
O33
O34
Ovar.tlse51
Ovar.tlse52
O1v(A681G&C1054T)

Akans(n = 137)
26
26
0
3
16
10
26
0
0
5
1
6
1†
0
1
10
2
1
0
0
0
0
2
1†

Berbers(n = 78)
22
40
4
1
6
1
1
0
2
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0

Basques(n = 220)
126
65
2
4
1
0
1
0
14
1
0
0
1*
3
0
0
0
0
0
0
2
0
0
0

Putien(n = 94)
55
39
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

Fujiou(n = 86)
44
42
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

Cayapas(n = 74)
37
31
0
1
1
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

Aymaras(n = 126)
34
75
0
0
0
0
0
15
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0

* All individuals were studied for exon 6 and exon 7 polymorphism. Individuals bearing representative alleles were studied by cloning andsequencing of both alleles they have. When DNA samples were still available, other individuals were studied by direct sequencing of intron 6.
† No intron 6 sequence was available for these alleles.

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Fig. 3. Schematic representations of five human recombinant alleles. The most probable parental alleles are shown by graphic codes. A recombinant allele described in the
text (Ovar.tlse20) is not included in the figure because one of the two parental alleles
was not sampled in the individuals studied. The possible recombination points flankthe black boxes, which correspond to regions where the two parental alleles are identical. *Inactivating mutation.

allele O03 differs most from a simple
recombinant by a single substitution
at e802, suggesting a more ancient
occurrence of recombination than forother recombinant alleles. In fact, datapreviously reported from upstream ofexon 6 demonstrated that O03 must be
considered as a mosaic of parts of fouralleles (A101, B101, O01, and O02).14 Discarding recombinant alleles from theanalysis, three major allelic lineages(A101-O01, B101, and O02) are observedin the network. Substantial differences
between O01 and O02 alleles werealready noted from comparison of
cDNA sequences, which is confirmedwith the information of intron 6.2
The proportion of nucleotide differences among these three major alleliclineages goes from 0.009 to 0.012 in
the 2-kb region analyzed. The genomic
average difference between human andchimpanzee was recently estimated tobe 0.0123.31 If we assume divergence
between human and chimpanzee to be
6 million years, the average evolutionaryrate becomes 1.0 ¥ 10-9 per site per year
for the whole genome. Under the roughassumption of constancy of the evolutionary rate, the diversification of thethree major human ABO alleles can be
estimated at 4.5 to 6.0 million years
ago. This unusually large divergencetime estimate is consistent with thatobtained through cDNA sequence
data.26DISCUSSION

Fig. 4. Phylogenetic network of 26 human ABO alleles. 1 nuc. dif. = one nucleotide
difference. Numbers in filled circles correspond to recombinant alleles. 3¢¢(e526-) = 3¢¢
end of the allele after position 526 in exon 7; 3¢¢(i446-) = 3¢¢ end of the allele after position
446 in intron 6; 5¢¢(–i1013) = 5¢¢ end of the allele before position 1013 in intron 6; and5¢¢(–i235) = 5¢¢ end of the allele before position 235 in intron 6. E297* means that mutation at this position for allele Ovar.tlse51 cannot be represented correctly in the network because the reverse mutation is observed at the same position between the root
and A101 group of alleles.

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Until now, in all populations studied
so far, alleles O01 and O02 are present
although in variable proportions. Apartfrom these two main O alleles, many
new alleles have been described, someof them first reported here thanks to theinformation provided by intron 6.
Among the infrequent alleles, even if
some have been reported in a single
population, they cannot be considered
population-specific. For example, allele
O26 (Ovar.tlse02) was originally foundonly in Basques but was later reported
in a German population.14 There is a lackof geographic structure owing to their
low frequency (drift therefore playing a

EVOLUTION OF HUMAN O ALLELES

major role in shaping the frequencies), the small sample
size for most of the studies, and the very deep roots of the
O branch in the ABO gene genealogy, much deeper than
the genealogy of the present human populations.32
It is interesting to note that, as in Iwasaki et al.,33
among the two Han Chinese populations studied here, we
found only two O alleles: O01 and O02. Using simple binomial sampling distributions, it can be shown that the
maximum frequency of an unobserved allele with a given
confidence level a (such as 0.05) in a sample of size N is p
= 1 – a1/N. Then, the maximum frequency at which alleles
other than O01 or O02 can exist is 0.062 for the Putien
population (n = 47) and 0.067 for Fujiou (n = 43). If pooled,
the maximum frequency drops to 0.033. This is highly surprising because even in Amerindians (Cayapas from Ecuador and Aymaras from Bolivia), who have lost A and B
alleles, the number of O alleles is at least 5 or 6, and O
alleles other than O01 and O02 add up to 0.11 and 0.135,
respectively.12 These results can be explained by a highgenetic homogeneity of some of the Han populations
owing to a founding effect, and the difference with theAmericas reflects the complex pattern of settlement of thiscontinent that does not justify considering any extant
Asian population as being very similar to the Amerindians
as if they were the “source” for American settlement. Somealleles found in the Amerindians, like O11 (O1v(G542A)), havenot been found in Asians, suggesting a recent and American origin of the allele (after 35,000 before present; see
Cavalli-Sforza and Feldman32).
In this study, we extended the characterization of O
alleles by sequencing intron 6 of all alleles encounteredin eight human populations. To compare the variability offunctional alleles we also cloned and sequenced someindividuals of A, B, or AB phenotypes. The description of
the intron 6 sequence has allowed some previous alleles
to be split into suballeles, but more interestingly, has given
a deeper insight into the origin and genetic relationship ofalleles. Two types of mechanism are involved in the diversification of O alleles: interallelic exchanges and point
mutations. From a phylogenetic perspective, if one allele
sequence is strictly equivalent to the recombination of thesequences of two alleles found nowadays in human populations, this is compatible with a recent recombination
event. Many examples of such recent recombinant alleles
have been reported8,11,26,28-30,34,35 and four new cases arecharacterized in this study (O1v-B.tlse14, O1v-B.tlse13,
Ovar.tlse20, and Ovar.tlse51). In contrast, if the potential
recombinant allele exhibits point mutations when compared to the theoretical parental alleles, it is compatible
with either an ancient recombination event or, less likely,
with a recent recombination as yet unsampled alleles, butwith recombination involved in any case. This corresponds to Ovar.tlse20.
It should be noted that the high divergence among
ABO alleles facilitates the detection of recombinant alleles(such as recombinants between A101 or O01 and O02 or
between or between B101 and A101 or O01) and, furthermore, allows the region where the putative crossover event
took place to be narrowed down to tens of bases. Obviously, recombination between highly similar alleles in
the analyzed region (such as O01 and A101) cannot be
detected. In the recombinants described here the parental
alleles have been detected (Fig. 3), showing that therecombination events do not cluster in particular spots,
even though a role in recombination for Ki and Ki-likesequences in intron 6 had been suggested.8,24 Recombination as a generator of variability in the ABO should not be
exceptionally high, because the local recombination rateis slightly under twice the genomewide average according
to the recent genetic map36 where recombination rates
were estimated along the genome by comparing genetic
to physical distances for a large number of microsatellite
markers. ABO lies 200 kb upstream from one such marker,
namely D9S754, around which the estimated recombination rate is 1.07 cM per million bases; for the 1875-bp
stretch we sequenced, that would translate to a recombination rate of 2.01 ¥ 10-5 per meiosis. It should also be
noted that the recombinant alleles could result either
from nonconservative interallelic exchange (crossingover) or from conservative interallelic exchanges throughgene conversion.
If one excludes O03, all human nonfunctional O alleles observed in the eight populations we studied share thesingle-nucleotide deletion at position 261 of exon 6. It ishighly improbable that such a deletion occurred at exactly
the same position more than once during human evolution especially because it is not located in a high mutation
rate context, like a stretch of poly(G). As a matter of fact,
point deletions are rarely observed among human alleles:
among all human ABO alleles reported so far, only one,
Ovar.tlse20, has a point deletion in intron 6, seven alleles(A201,37 A206 (Av1),10 A302,38 Aw01, Aw03, Aw05,6 andAw0714) share one point deletion at nucleotide 1060 inexon 7, and allele Ael0313 has a point deletion at nucleotide
804 in exon 7. If there is a common origin for deletion
D261, then it remains to be explained how this mutation
is observed in such divergent alleles as O01 and O02 (seeFig. 4). Allele O02 is clearly the more ancient human allele
harboring the single nucleotide deletion, because it is very
different from the extant functional alleles A101 and B101
and it must have been an ancient and independent lineage. Since then, allele O02 would have accumulated
many mutations without functional impact because it is anull allele. In that context, all other alleles sharing thisdeletion with O02 must be more recent and could havebeen transferred from the O02 to the O01 lineage either byrecombination between O02 and A101 or by conversion
between the two previous alleles. Nevertheless, upstreamof exon 6, the sequences of O01 and O02 are quite divergent,14 suggesting that gene conversion is the most likely
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ROUBINET ET AL.

mechanism for the presence of D261 in O01. The transferof the deletion to the A101 allele created an intermediate
allele that has not been found in any population studied
so far. Then, a point mutation in intron 6 (i784) appearedin this intermediate recombinant allele to create O01.
It remains to be explained, however, how the newrecombinant O01 allele frequency increased, leading thisallele to coexist with allele O02 in a wide range of humanpopulations.10-12,29 Because O01 and O02 are nonfunctional and therefore have identical selective values, only
chance can explain the persistence of the two alleles in all
human populations and both had to be present in themain human dispersal events, including the one out of
Africa. In spite of the fact that balancing selection is often
invoked to explain the maintenance of human ABO polymorphism, the persistence of the two main O silent allelesin humans must be explained by genetic drift. Because thenumber of differences between O01 and O02 is comparable to the number of differences between A101 and B101,
we may speculate that the persistence of A/B polymorphism is also due to chance, because the number ofsubstitutions in the three main lineages (A, B, and O) issimilar. In this context it should be recalled that chimpanzees lack the B allele, whereas gorillas lack both A and O
alleles.39 Moreover, the two O alleles characterized in
chimpanzees40 are totally different from those observedin humans, indicating that O silent alleles appeared
independently during the evolution of humans andchimpanzees.
It is thus clear that, even if lineage O01 is ancient, itcannot be considered as a founding lineage in humans,
contrary to the description of Seltsam et al.14 This is also
the case of allele O03, which is not among the deepest
branches of the ABO gene genealogy. As first suggested by
Irshaid and coworkers27 and Seltsam and colleagues14 it
is a complex recombinant, making O03 necessarily more
recent than the other alleles. Moreover, it has accumulated
less variation, has generated fewer alleles, and has a more
restricted geographic distribution than A101, B101, O01,
or O02. In fact, the position of O03 in a neighbor-joining
tree or in other representations (see Fig. 4) should not beread as the result of a long process of mutation accumulation, but rather as an artifact caused by recombination.
The evolution of the ABO system, beyond the dynamics involving mutation and recombination from the threemain branches, shows extremely long roots in the humanlineage (5-6 millions years), around the same age as timeof separation with chimpanzee lineage.
ACKNOWLEDGMENTS
The authors are very grateful to I. Desmoulins (EFS Aquitaine
Limousin, Site de Biarritz, France), A. Chaventré (University Bordeaux II, Bordeaux, France), A. Pitte (Abidjan, Ivory Coast), R.
Calderon (Universidad del Pais Vasco, Bilbao, Spain), G.F. De Ste714

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fano (University Tor Vergata, Rome, Italy), A. Baali and M.
Cherkaoui (University Cadi Ayyad, Marrakech, Morocco), and M.
Villena (Instituto Boliviano de Biologia de Altitudes, La Paz,
Bolivia) for providing human blood or DNA samples. The authors
are also indebted to W.W. Socha and G. Dubreuil for providing
primate samples. We thank Marianne Dutaur and Pierre Tisseyre
for their technical assistance.

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