Hum Genet (1996) 97 : 777-783© Springer-Veflag I996

Kenichi Ogasawara • Makoto Bannai • Naruya Saitou
Ryuichi Yabe • Kenichi Nakata • Michiko Takenaka
Kiyoshi Fujisawa • Makoto Uchikawa
Yoshihide Ishikawa • Takeo Juji • Katsushi Tokunaga

Extensive polymorphism of ABO blood group gene:
three major lineages of the alleles for the common ABO phenol3,pes
Received: 3 July 1995 / Revised: 1 September 1995

Polymorphism of the ABO blood group gene
was investigated in 262 healthy Japanese donors by apolymerase chain reactions-single-strand conformation
polymorphism (PCR-SSCP) method, and 13 different alleles were identified. T h e n u m b e r of alleles identifiedin each group was 4 for A I (provisionally called
ABO*A101, *A102, *A103 and *A104 according to theguidelines for human gene nomenclature), 3 for B(ABO*B101, *B102 and *B103), and 6 for O (ABO*O101,"O102, "O103, "O201, * 0 2 0 2 and *0203). Nucleotide
sequences of the amplified fragments with different SSCP
patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could beclassified into three major lineages, *A/*O1, *B and *02,
In Japanese, *A102 and *B101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles,*O101 (43%) and "O201 (53%), were observed. These
results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be
advantageous indicators for population studies.

Abstract

Introduction
The gene that determines ABO blood group, and which
encodes a specific glycosyltransferase, was recently
cloned and sequenced by Yamamoto et al. (1990a,b). As aresult of these observations, several investigators have developed ABO genotyping methods that use polymerase
chain reaction-restriction fragment length polymorphism(PCR-RFLP) analysis (Lee and Chang 1992; O ' K e e f e andDobrovic 1993; Stroncek et al. 1995) and allele-specific
PCR (Ugozzoli and Wallace 1992). Fischer et al. (1992)
applied the PCR-RFLP method to the diagnosis of the acquired B status. Subsequently, Yamamoto et al. (1992,
1993a--d) determined the sequences of alleles for weak
antigen groups, such as A 2, A 3, B 3, cis-AB, A x and B (A), as
well as another type of O allele.
From these observations, it seems that the polymorphism in the ABO blood group gene remains to be investigated. We have therefore developed a PCR-single-strand
conformation polymorphism (SSCP) method for analyzing the extensive polymorphism of the ABO blood group
gene.

Materials and methods
DNA samples
K. Ogasawara ([I~) - M. Bannai - R. Yabe - K. Nakata
M. Takenaka - K. Fujisawa
Japanese Red Cross Tokyo Metropolitan Blood Center,
1-26-1 Kyonan-cho, Musashino-shi, Tokyo 180, Japan
Tel.: (81)422-32 19 95; Fax: (81)422-32 26 85

Genomic DNAs were prepared from peripheral blood leukocytesof 262 unrelated Japanese voluntary donors by standard phenolchloroform extraction (Sambrook et al. 1989). The donors werecomposed of 74 blood group At, 73 group B, 61 group AIB and 54
group O individuals.

N. Saitou
Laboratory of Evolutionary Genetics,
National Institute of Genetics, Japan

Analysis by PCR-SSCP

M. Uchikawa • Y. Ishikawa • T. Juji • K. Tokunaga
Japanese Red Cross Central Blood Center, Japan
K. Tokunaga
Department of Human Genetics,
Graduate School of International Health,
University of Tokyo, Tokyo, Japan

PCR conditions and sequences of all primers are shown in Tables
1 and 2. PCR amplifications of four fragments (I IV) performed
with 35 amplification cycles in 10 ~tl of PCR reaction mixture(Bannai et al. 1994a). As shown in Fig. 1, PCR fragments I-IV
cover the last two exons (exons 6 and 7) (Bennett et al. 1995; Yamamoto et al. 1995) of the ABO gene at nucleotide positions
240-374, 375-564, 526-855 and 845-1065, respectively. Total

778
length of exons 6 and 7 is 826 bp (78% of the coding sequences)whereas that of exons 1-5 is 239 bp (22%).
SSCP analysis was performed essentially as described previously (Bannai et al. 1994a), using a 12.5%-15% polyacrylamide
gel with or without 5% (by vol.) glycerol. Briefly, 1 lu[ of amplified DNA solution was mixed with 7 ~1 of denaturing solution,
heated at 95°C for 5 rain, and immediately chilled on ice; I ~1 ofthe mixture was subjected to polyacrylamide gel electrophoresis.
The separated single-stranded DNA fragments in the gel were visualized by silver staining.

T a b l e 1 Nucleotide sequences of primers used in this study
Name

Sequence

Sense primers
ABO-I
ABO-3
ABO-5
ABO-7
ABO-9
GS-AB
GS-O
GS-AO

5"-CATGTGACCGCACGCCT-3"
5"-CCGTCCGCCTGCCTTGCAG-3"
5"-CAGCTGTCAGTGCTGGAGGTG-3"
5"-AAGAGGTGCAGCGGCTCACCA-3"
5"-GCCAAAGGTGCTGACACCG-3"
5"-AGGAAGGATGTCCTCGTGGTGG-3"5"-AGGAAGGATGTCCTCGTGGTA-3"
5"-CCATTGTCTGGGAGGGCATA-3"

Group-specific PCR

Antisenseprimers
ABO-2
ABO-4
ABO-6 a
ABO-8
ABO-105"-TCGGCCACCTCACTGACTTA-3"
5"-AAGTCACTGATCATCTCCAT-3"
5"-CCGTTGGCCTGGTCGACCATCATGGCCTG-3 ~5"-AGCCCTCCCAGAGCCCCTGGCA-3"
5"-TGCTAAAACCAAGGGCGGGA-3"" Identical to FY-2 reported by Yamamoto et al. (1990a)

Table 2Sense
primer

ABO-I
ABO-3
ABO-5
ABO-7
GS-AB
GS-O
GS-AO
ABO-3
ABO-9

As shown in Table 2, three sets of primers were used for group-specific PCRs: GS-AB and ABO-10 for groups A and B, GS-O andABO-10 for group O. and GS-AO and ABO-10 for a part of the A
and O alleles. These sense primers (GS-AB, GS-O and GS-AO) arelocated within fragment 1, and the 3"-terminal of the antisense primer(ABO-10) is located t9 bases from the 3"-terminal of fragmen~ IV(Fig. l). Thus, these amplified fragments include fragments It
through IV completely. The group-specific PCRs were performed
under the same conditions as PCR-SSCP except that 5% (by vol.) dimethyl sulfoxide (DMSO) was added to the PCR mixture,

Polymerase chain reaction (PCR) and single-stand confirmation polymorphism (SSCP) conditions for each primer pair
Antisense
primer

ABO- 2
ABO- 4
ABO- 6
ABO- 8
ABO- 10A BO- t 0
ABO- 10
ABO- 8
ABO- 10

Product
length(bps)

PCR annealing
temperature
(°C)

187
228
380
269
1900
1900
1900
737
2500

60
58
66
65
61
63
61
65
63

SSCP

Remarks

Gel concentration(%)

Electrophoretic
temperature (°C)

15
13
13
12.5*
. .

12
30
26
30

-

.

.

Fragment t
Fragment 11
Fragment Ill
Fragment IV
Group-specific PCRGroup-specific PCRGroup-specific PCR
Template for direc!
sequencing.

-. . . . . . . .
--* With 5% glycerol

Fig. 1 Positions of primers andamplified fragments in the
ABO gene. Numbers indicate
the nucleotide positions of the5"- and 3"-terminals of the last

240

374

375

1066

lExon 7 1
ABO-1

ABO-2

ABO-3

ABO-4

tWO e x o n s

Fragment I

Fragment 11
ABO-5

ABO-6
4Fragment ]]1
ABO-7

ABO-8
4P
Fragment IV
ABO-10

ABO-9

ABO - 10

GS-ABIO/AO

ABO-3

ABO-8

779
Direct sequencing

Table 3 SSCP patterns for each allele

DNA templates for cycle-sequencing were produced by the following PCRs. For samples possessing only a single allele, fragments (approximately 2.5 kb including fragments I through IV and
two introns) were amplified using the generic primer pair ABO-9
and ABO-10. For samples possessing two different alleles, a 737bp fragment (fragments II through IV) was amplified by the primer
pair ABO-3 and ABO-8 using the group-specific PCR products as
templates. These amplified DNAs were purified as described previously (Bannai et al. 1994b) using a 1% low-melting-temperature
agarose gel (BRL, Gaithersburg, Md., USA). The purified DNAs
were then directly sequenced at least twice for each direction by acycle-sequencing method with the same primers as used for PCRSSCP, using an automated DNA sequencer (373A; Applied Biosystems, Foster City, Calif,, USA).

AlleleProvisional classification of ABO alleles
The observed alleles were provisionally named according to theguidelines for human gene nomenclature (Shows et al. 1987).
Phylogenetic analysis

e

la/Ia

lb/Ib

Id/Id

FI

F II

F III

F IV

Ia
la
Ia
Ib
Ib
Ib
Ib
Ic
Ic
Id
Id
Ic
Id

IIa
IIb
IIc
IIa
Ild
IId
IId
IIa
IIa
IIa
IIa
IIa
IIa

IIIa
IIIa
IIIa
IIIa
IIIb
IIIb
IIIc
Ilia
IItd
IIIa
IIIe
IIIe
IIIf

IVa
IVa
IVa
IVa
IVb
IVa
IVb
IVa
IVa
IVa
IVa
IVa
IVa

Results

The maximum parsimony method (Fitch 1977) and the phylogenetic network method (Bandelt 1994) were used for delineating therelationship of alleles. The phylogenetic network method is closely
related to the maximum parsimony method, although it may produce networks instead of trees. This method can be considered to
be a generalization of a discordancy diagram (Fitch 1977). The
computer program PAUP 3.1.1 (Univ. Illinois National History
Survey) was used in the parsimony analysis, while the phylogenetic network was produced manually.

a

ABO*A 101
*A 102*A 103*A104
ABO*B 101
*B 102*B 103
ABO*O 101
*O 102*O 103
"O201
*0202
*0203

SSCP pattern

Ic/Ic

b

lib/lib IId/IId IIa/IIa IIc* IIc/Ild

IIIa/IIIa llId* IIIc* IIIb/llIb IIIe/IIIe IIIf*d

IVa/lVb IVb/lVb

Fig. 2a-d PCR-SSCP analysis of the gene determining the ABO
blood group, a Fragment |, four different homozygous samples; bfragment II, three different homozygous samples and one single allele (IIc*) obtained by group-specific PCR from a heterozygous
sample (lIc/IId); e fragment lIl, three different homozygous samples and three different single alleles (IIIc*, IIId*, and IIIf*) obtained by group-specific PCRs; d fragment IV, two different homozygous samples

A B O alleles identified by P C R - S S C P
Genomic DNAs from 262 donors with known A B O phenotypes, which were determined by serology, were examinedby PCR-SSCP. A typical SSCP pattern obtained from asample of an individual possessing a single allele showed
two bands, corresponding to sense and antisense strands,
and the fragments derived from two different alleles
showed four bands (Fig. 2). In a few cases, three-band patterns were observed, although the samples carried single
alleles (Fig. 2a). This was probably due to electrophoresisof a large amount of DNA, or to equilibration o f two different conformations of single-stranded D N A fragments.
The numbers o f different SSCP patterns observed for eachfragment were four for fragment I, four for fragment II, six
for fragment III, and two for fragment IV. The alleles wereidentified based on the combination of the SSCP pattems
of these four fragments. When SSCP patterns of fragments
I - I V showed only a single pattern with two bands, thesample was judged to be from a homozygote carrying ac o m m o n allele. Forty-four out of the 262 samples were
judged to be homozygotes for c o m m o n alleles and 195
samples as heterozygotes possessing these c o m m o n alleles.
U n c o m m o n alleles were identified by subtracting thepatterns of the c o m m o n alleles from those observed in theremaining 23 heterozygous samples. These rare alleles
were further analyzed to confirm the combination o f fragments I - I V using nested PCR. By group-specific PCRs,
only one allele out of the two in heterozygotes could bespecifically amplified (first PCR); with the primer pairG S - A B / A B O - 1 0 for groups A and B, G S - O / A B O - 1 0 forgroup O, and G S - A O / A B O - 1 0 for a part of the A and O
alleles. The D N A fragments obtained from the single allele including fragments I I - I V were then subjected to
P C R - S S C P analysis for each fragment (second PCR).
Thus, [he S S C P patterns o f these fragments showed only
two bands as demonstrated in Fig. 2b, c.

780
As shown in Table 3, the SSCP patterns observed ingroup A ~ were provisionally called Ia and Ib for fragment
I, IIa, IIb and IIc for fragment II, IIIa for fragment III, andIVa for fragment IV. Consequently in group A ~, we identified four different alleles, A B O * A I 0 1 , *A102, *AI03
and *A104, based on the combination of SSCP patterns.
Among them, ABO*A101 and *A102 were observed in
the homozygous state.
In group B, the observed SSCP patterns were Ib forfragment I, lid for fragment [I, IIIb and IIIc for fragment
III, and IVb for fragment IV. Among them, IId, IIIb andIIIc were exclusively observed in group B. Consequently
in group B, we identified three different alleles, withABO*B 101 being the predominant one.
In group O, the observed SSCP patterns were Ic and Id
for fragment I, IIa for fragment II, IIIa, llId, IIIe and IIIf
for fragment II], and IVa for fragment IV. Among them, Ic
and Id were restricted to group O. Two of the six O alleles, ABO*O101 and "O201, were commonly observed.
In total, we identified 13 different alleles. Moreover,
PCR-SSCP analysis of fragments I and II, or fragments I
and III, allowed us to determine the common ABO genotypes: SSCP patterns Ic or Id for O, IId and IIIb or IIIc for
B, and other patterns of fragments I - I I I for A t.

Fig.3 Schematic comparisonof nucleotide and deduced
amino acid sequences of the
ABO alleles identified by
PCR-SSCP. Only the differences from the nucleotide andamino acid sequences of*AI01 are indicated. An entirely different amino acid sequence in O alleles (m) resulting from frame-shifting caused
by a single base deletion.
*A101, *B101, *Ol01 and"O201 have sequences identical to previously described
ABO alleles (Yamamoto et al.
1990a,b)

Nucleotide sequences of ABO alleles
All of the D N A fragments that were distinguished fromeach other by SSCP analysis were sequenced in order toreveal any identity with previously reported sequences.
Figure 3 shows a summarized schema of the sequencing
results.
In group A I, ABO*A101 had an identical sequence tothat of the cDNA clone FY-66-1 described by Yamamoto etal. (1990b). ABO*A102 had a single nucleotide substitution at codon 156 as compared with ABO*AI01:
ABO*A102 has CTG coding for leucine, while ABO*A101
has CCG coding for proline. The same substitution had already been observed in FY-59-5 (Yamamoto et al. 1990a),but the sequence of this clone differed by a three-base deletion at nucleotide positions 240-242 from that of the otherclones (Yamamoto et al. 1990b) and the present alleles including ABO*A102. [Concerning the nucleotide numbering system in the ABO gene, the previous numbers (Yamamoto et al. 1990a, b) are different from the more recent
numbers (Yamamoto et al. 1992, 1993a-d) used in thisstudy.] ABO*A103 had a single synonymous substitution at
nucleotide position 564 as compared with ABO*A102.
ABO*A104 and *A101 differed by a single synonymous
substitution at position 297 (G and A, respectively).

261
240

297

467
C

526
C

664
579
CT

I

657 703646 681 721
TCGGC
II

*.4101IJI~ 6 829771 8 ~
CCGG
I

I!

930
G

I

P;o

A;g

&y'

L~a'GJy

156

176

235

266 268

AC

f

T
I

*,4102*.4103

L;u
I

T

T

I

I

L;u
G

*AI04

[

*B101

I

*B102

!

G

G

T

A

I

I

1

I

T

Ser
A

Me~t'Ala
AC

~y

sir

Met'Ala

G

A

AC

~y

I

G

G

I

t

G

*B103

H

|

I

t

Gly

iI

T
~G

G

Jl

I

AG G

"0201
ZXG

i1
Z~G G

*0203

Met'Ala

STOP

ZxG

*0202

II

sir

r

"0102
"0103

I

STOP

AG

"0101

II

I

STOP

r
STOP

A

A

r

T

I

STOP

A

A
I

I
STOP

A

T

T

A

I

I

I

I

I

......

1065

In group B, ABO*B101 had an identical sequence tothat of a previously described B allele (Yamamoto et al.
1990b), whereas ABO*B102 and *B103 had synonymous
substitutions that differed from *B101 at positions 930 (Ato G) and 657 (T to C), respectively.
In group 0 , all six alleles shared a single nucleotide
deletion (G) at position 261, resulting in a shift in thereading frame that leads to the appearance of a stop codonat positions 353-355. These six alleles could be classified, Fig.4 A phylogenetic network for the 13 ABO alleles (denoted byaccording to the substitutions and phylogenetic network full circles). Open circles indicate intermediate states. Numbers indescribed below, into two subgroups, ABO*Ol and "02. circles correspond to the following polymorphic sites: 1 = 261,2 =ABO*0101 was different from ABO*0201 by five nu- 297,3 =467,4=526,5=564,6=579,7 =646,8 =657,9=681,
10=703, 11 =721, 12=771, 13=796, 14=803, 15=829, 16=
cleotide substitutions at positions 297, 646,68 1, 77 1, and 930
829, and both alleles were identical to the previously described 0 alleles (Yamamoto er al. 1990b, 1993~).
ABO"0102 and "0103 had a one-nucleotide substitution that of the A' allele described by Yamamoto et al. (1992),
compared with ABO*0101 at positions 579 (T to C) and its estimated frequency in the Japanese population was
297 (A to G), respectively. Similarly, AB0*0202 and much lower than that of ABO"A102 (approximately one*0203 had a one-nucleotide substitution compared with fifth). Within group B, ABO*B101 was the predominant
ABO"0201 at positions 297 (G to A) and 721 (C to T), allele at a frequency of 97%. In contrast, within group 0,
respectively.
both ABO*0101 and "0201 were common with similar
Thus, 9 of the 13 alleles (ABO"A102, *A103, *A104, frequencies (43% and 53%, respectively).
*B102, *B103, "0102, "0103, "0202 and *0203) were
newly sequenced ABO alleles, but none of their nucleotide substitutions, except for position 467 of *A102 Phylogenetic tree of ABO alleles
and *A103, lead to amino acid changes.
We first applied the maximum parsimony method to thesequence data of the 13 ABO alleles for reconstructing theEstimation of allelic frequencies
phylogenetic tree. Thirty-six equally parsimonious trees
were produced using PA'UP 3.1.1 with the branch-andWe selected samples from a total of 262 healthy Japanese bound option. When interior branches with length zerodonors, in which 74 A,, 73 B, 61 A,B and 54 0 phenotypes were ignored, however, the number of distinct tree topolowere included, and ABO allele frequencies in each group gies was reduced to 16 (trees not shown). Clearly, thewere examined by PCR-SSCP analysis (Table 4). Within maximum parsimony method is not appropriate for delingroup A', ABO*A102 was most common at a frequency of eating the complex nature of the polymorphism of the
83%. Although ABO*A101 had an identical sequence to ABO alleles.
We also used the phylogenetic network method, andthe resulting single network is presented in Fig.4. There
are three rectangles in that network, indicating the exisTable 4 Allele frequencies in ABO systemtence of mutually incompatible sites. For example, theProportion within rectangle consisting of three B alleles is produced because
Alleleeach groupthe nucleotide configuration for site 8 (position 657 in
Fig. 3) is inconsistent with that for site 16 (position 930 in
ABO*AlOl
23
15.4
Fig. 3). Either site is assumed to experience parallel sub*A102
123
82.6
stitutions. In any case, all the 16 equally parsimonious
*A103
1
0.7
trees can be created from this single network.
*A104
2
1.3
Total

149

100.0

ABO*B101
*B102
*B103
Total

138
3
142

97.2
2.1
0.7
100.0

ABO*0101
*0102
*0103
ABO*0201
*0202
*0203
Total

100
3
1
123
5
1
233

42.9
1.3
0.4
52.9
2.1
0.4
100.0

1

Discussion
Since the molecular structure of the gene determining the
ABO blood group has recently been elucidated (Yamamoto et al. 1990a,b), several DNA typing methods including PCR-RFLP analysis and allele-specific PCR havebeen described. These methods might be applicable inforensic science (Lee and Chang 1992; Matsuki et al.
1994; Fukumori et al. 1995), laboratory diagnosis (Fischer
et al. 1992) and population studies (Grunnet et al. 1994;
Franco et al. 1994) with limited ABO alleles as the sub-782
jects. A l t h o u g h P C R - R F L P and allele-specific P C R methods are useful for detecting variations in the recognition
sequences o f certain restriction e n z y m e s or specificprimers, they cannot necessarily be used to detect alleles
with unknown substitutions in the a m p l i f i e d D N A fragments. Recently, Johnson and H o p k i n s o n (1992) described four different O alleles, as well as two B and one
A allele from unrelated E u r o p e a n individuals b y m e a n s o f
denaturing gradient gel electrophoresis, but no sequence
analysis was performed. F r o m their observations and apersonal c o m m u n i c a t i o n (K. K o b a y a s h i ) , the n u m b e r o f
different alleles in the A B O b l o o d group system is considered to be much m o r e than generally anticipated.
In this study, we applied a P C R - S S C P m e t h o d to detect
different A B O alleles. This m e t h o d enabled us to detect
point mutations at various positions (Orita et al. 1989) in
the amplified fragments, and it can be used to d e t e r m i n e
the A B O g e n o t y p e s as well as to detect undescribed A B O
alleles. In this P C R - S S C P analysis, we identified 13 different alleles and the numbers o f alleles in groups A ~, B
and O were 4, 3 and 6, respectively. F o u r o f them,A B O * A 1 0 1 , *B101, *O101 and "O201, had sequences
identical to those o f the previously d e s c r i b e d A B O alleles( Y a m a m o t o et al. 1990a, b). T h e o t h e r nine a l l e l e s ,A B O * A 1 0 2 , *A103, *A104, *B102, *B103, "O102,"O103, * 0 2 0 2 and * 0 2 0 3 were n e w l y observed.
A B O * A 1 0 2 has an identical sequence to that o f
A B O * A 1 0 1 except for a single base substitution at nucleotide position 467 (C to T). A B O * A 1 0 3 and * A 1 0 4
have o n l y one or two nucleotide substitutions c o m p a r e dwith A B O * A I 0 1 or *A102, and thus these 4 A B O * A alleles m a y belong to a single e v o l u t i o n a r y lineage.
Y a m a m o t o et al. (1990b) d e s c r i b e d a B allele withseven nucleotide substitutions c o m p a r e d with an A ~ allele,and four o f them lead to a m i n o acid changes that m a y be
responsible for the specificity o f A~ or B transferase.
T h e s e four nucleotide substitutions were also o b s e r v e d in
the three B alleles o b s e r v e d in the present study.
A B O * B 102 and *B 103 have only one nucleotide substitution c o m p a r e d with A B O * B 1 0 1 . Thus, these three
A B O * B alleles b e l o n g to a single e v o l u t i o n a r y lineage,
which is different from the A B O * A lineage.
In group O, we identified six different alleles, all o fwhich shared a single nucleotide deletion at position 261.
In contrast, in the Japanese population s a m p l e s e x a m i n e d
to date, we could not find another type o f O allele n a m e d
02 (Yamamoto et al. 1993d; Franco et al. 1994). ABO*O101
has an identical sequence to that of A B O * A I 0 1 except fora single nucteotide deletion. A B O * O 1 0 2 and *O 103 have
one nucleotide substitution c o m p a r e d with A B O * O I 0 1 .
Thus, these three alleles m a y belong to the s a m e evolut i o n a r y l i n e a g e as A B O * A . In contrast, A B O * O 2 0 1 has
5 nucleotide substitutions c o m p a r e d with A B O * O 1 0 1 .
Moreover, A B O * O 2 0 2 and * 0 2 0 3 have one nucleotide
substitution relative to A B O * O 2 0 1 , and thus these threealleles m a y belong to another lineage, which is different
from the *A or *B lineage.
T h e s e results indicate that the Japanese A B O alleles
are d i v i d e d into three m a j o r lineages, * A / * O I , *B and* 0 2 . P h y l o g e n e t i c network analysis o f these A B O alleless u p p o r t e d the a b o v e hypothesis.
O u r P C R - S S C P m e t h o d m a y be useful for the elucidation o f the variety o f the A B O gene as well as the establishment of A B O genotyping. Furthermore, the method
w o u l d be a p p l i c a b l e for identifying alleles that d e t e r m i n e
uncommon phenotypes.
Acknowledgement We thank Mrs. Hiroko Ogata for her technical assistance in the preparation of the genomic DNAs.

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