Tissue Antigens ISSN 0001-2815

Determination of HLA-A, -C, -B, -DRB1 allele and haplotype
frequency in Japanese population based on family study
N. Ikeda1 , H. Kojima1 , M. Nishikawa1 , K. Hayashi1 , T. Futagami1 , T. Tsujino1 , Y. Kusunoki1 , N. Fujii1 ,S. Suegami1 , Y. Miyazaki1 , D. Middleton2 , H. Tanaka1 & H. Saji1
1 HLA Foundation Laboratory, Kyoto, Japan
2 Transplant Immunology, Royal Liverpool University Hospital Trust and University of Liverpool, Liverpool, UK

Key words
allele frequency; family study; haplotype
frequency; human leucocyte antigen;Japanese population
Correspondence
Hiroh Saji
HLA Foundation Laboratory
2F#1 Kyoto Research Park
134, Chudoji Minami-machi
Kyoto 600-8813
Japan
Tel: +81 75 313 5201
Fax: +81 75 313 5202
e-mail: hla@hla.or.jp
Received 18 August 2014; revised 14
January 2015; accepted 3 February 2015

Abstract
The present study investigates the human leucocyte antigen (HLA) allele and haplotype frequencies in Japanese population. We carried out the frequency analysis in 5824
families living across Japanese archipelago. The studied population has mainly been
typed for the purpose of transplant, especially the hematopoietic stem cell transplantation (HSCT). We determined HLA class I (A, B, and C) and HLA class II (DRB1)
using Luminex technology. The haplotypes were directly counted by segregation. A
total of 44 HLA-A, 29 HLA-C, 75 HLA-B, and 42 HLA-DRB1 alleles were identified.
In the HLA haplotypes of A-C-B-DRB1 and C-B, the pattern of linkage disequilibrium
peculiar to Japanese population has been confirmed. Moreover, the haplotype frequencies based on family study was compared with the frequencies estimated by maximum
likelihood estimation (MLE), and the equivalent results were obtained. The allele andhaplotype frequencies obtained in this study could be useful for anthropology, transplantation therapy, and disease association studies.

doi: 10.1111/tan.12536

Introduction

The human leukocyte antigen (HLA) gene family is characterized by extreme degree of genetic polymorphism and linkage
disequilibrium (LD). The varieties in polymorphism and LD
patterns of HLA gene family show a tendency to be uniquein each ethnic group (1, 2). HLA antigens have been known
to play an important role in immune responses. In hematopoietic stem cell transplantation (HSCT), HLA matching between
donors and recipients lowers the risk of graft rejection andgraft-versus-host disease (GVHD) (3, 4). Morishima et al. suggested that the genetic difference derived from HLA haplotype
is associated with acute GVHD in allogeneic HSCT (5). Therefore, HLA haplotype cannot be excluded from consideration
during donor selection because of potential contribution fromproteins encoded by non-HLA genes inherited with HLA genes.
Several studies reported analysis of HLA allele and haplotype frequency data in the Japanese population (6–9). However,these studies failed to present accurate and detailed information
due to the haplotypes estimation using software or small sample
size, or both. This necessitates developing a method which can
produce accurate and detailed gene distribution. The present
study aims to obtain a more exact and detailed HLA haplotype
distribution from 18,604 members of 5824 Japanese families,
252

whose HLA haplotypes were determined by descent. Our study
also attempts to determine the frequency of specific haplotypes,
C-B, A-B-DRB1, and A-C-B-DRB1, used in donor search. In
addition, it was ascertained whether the haplotype frequencies
estimated by maximum likelihood estimation (MLE) would beequivalent to the frequencies found in the present family study.
Materials and method
Subjects

A total of 18,604 members (including patients and normal
subjects) from 5824 families, distributed in all parts of Japan,
were enrolled for this study. Among these families, there were
patients, considered for transplantation, especially HSCT. The
families were divided into three groups (Table 1): (i) families
with both parents with one or more children, (ii) families with
one parent with one or more children, and (iii) families with noparents but having two or more children. The families withmore than two generations were counted as separate families.
Informed consent was obtained from all the participants of thisstudy by the clinicians who ordered HLA typing.
For comparing the haplotype frequencies obtained by family
study and using MLE software, unrelated 4500 people were
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tissue Antigens, 2015, 85, 252–259

N. Ikeda et al.

HLA allele and haplotype frequency in Japanese population

Table 1 Breakdown of the family structure

Parents
One parent
No parent

1+2

1

Number of children

Number of familiesa

A

B

1–5
1–8
2–7
Total

2077
1968
1779
5824

a/b

c/d

C

D

E

b/d

a/c

e/f

a

The families with more than two generations are counted as separate
families.

chosen at random from the total subjects of present study(18,604 members). They were genetically unrelated, because
one person was chosen from each of 4500 random chosen families. The overlaps of blood relationship with three or more generation were avoided in these families. The allele and haplotype
frequencies calculated from these 4500 people were very similar to the frequencies from the total subjects [allele frequencies(AF) data not shown].
Samples

DNA samples were obtained from peripheral lymphocytes or
buccal cells using a JetQuick® Blood & Cell Culture Kits
(GENOMED, Löhne, Germany) or QuickGene DNA Tissue
Kit (KURABO, Osaka, Japan) according to the manufacturer’s
protocols.
HLA allele typing

HLA (-A, -C, -B, and -DRB1) four-digit allele typing was
performed using Luminex 200 system (Luminex, Austin, TX)
and WAKFlow HLA Typing kit (Wakunaga, Hiroshima, Japan)(10–12). HLA alleles were assigned automatically using
WAKFLOW Typing software (Wakunaga, Hiroshima, Japan).
The primer sequences of WAKFLOW Typing kit are specificallydesigned to make allele determination easier in Japanese population, and by default the analysis with WAKFLOW Typing
software is based on the AF of the donors registered withJapan Marrow Donor Program (JMDP) which are available on
the website, www.bmdc.jrc.or.jp (9). Therefore, this method
can determine alleles with frequencies of 0.1% and greaterin the Japanese population. A few alleles which could not be
determined by this method as rare alleles were considered as
secondary; these alleles were determined using Luminex 200
system and LAB Type SSO kit (One Lambda, Los Angeles,
CA) assigned using the HLA Fusion software (One Lambda).
In brief, exon 2 for HLA-DRB1; exons 2 and 3 for HLA-A, -B,
and -C were amplified in these methods.
Haplotype determination

The haplotypes were determined by segregation. This study was
designed with the aim to assess genetic linkage with high certainty. Nevertheless, the results also included some partial haplotypes because of the possibility of one or more recombination© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tissue Antigens, 2015, 85, 252–259

2

F

G

H

a/f

a/e

c/f

A, mother 1; B, father 1; C, child 1-1; D, child 1-2
D, mother 2; E, father 2; F, child 2-1; G, child 2-2; H, child 2-3

1, family 1

2, family 2

1+2, family 1+2

ex). Haplotype identification results are assumed as below.
A has a/b. D has a/c.
a. A*24:02–B*52:01–DRB1*15:02
B has c/d. E has e/f.
b. A*33:03–B*44:03–DRB1*13:02
c. A*24:02–B*07:02–DRB1*01:01
C has b/d. F has a/f.
d. A*24:02–B*54:01–DRB1*04:05
D has a/c. G has a/e.
H has c/f.
e. A*02:07–B*46:01–DRB1*08:03
f. A*11:01–B*15:01–DRB1*04:06
Figure 1 Avoiding haplotype duplication. The extended haplotypes of aand c are redundant in families 1 and 2. In this case, six haplotypes werecounted as family 1 + 2.

in a family. The haplotype frequencies were calculated byusing haplotypes without taking into account the recombination. Thus, we counted the haplotypes of parents and not ofchildren because fathers and mothers are genetically unrelated,while the haplotypes created by recombination were those ofchildren only. Some haplotypes of parents whose children had
recombinant haplotypes could not been determined becauseof two patterns of their combinations. These haplotypes were
determined and counted as not less frequent but frequent haplotype phase. Specifically, assuming that four haplotypes are Hp1,
Hp2, Hp3, and Hp4, those frequencies are HF1, HF2, HF3, andHF4, and two estimable phases are ‘Hp1, Hp2’ and ‘Hp3, Hp4’,
HF1 was multiplied by HF2, HF3 was multiplied by HF4, andthe haplotypes of the estimable phase with larger product werecounted.
For the comparison to the result by MLE, the haplo.em program, which was evaluated by HAPLO.STATS (version 1.6.0)
software operated in the R language, was used (13–16). For
genetic markers measured on unrelated subjects with linkage
phase unknown, this program computes the maximum likelihood estimates of haplotype probabilities using the progressive
insertion algorithm that progressively inserts batches of lociinto haplotypes of growing lengths.
253

N. Ikeda et al.

HLA allele and haplotype frequency in Japanese population

Table 2 HLA-A, -C, -B, -DRB1 allele frequencies in Japana
HLA-A
serological
speciﬁcity
A1
A2

A203
A210
A3
A11

A24

A2403
A26

A29
A30
A31
A32
A33
A34
A66
A68
Null
Undeﬁned

HLA-A
allele

HLA-C serological
GF (%)
speciﬁcity

A*01:01
0.401
A*02:01
11.620
A*02:06
9.081
A*02:07
3.461
A*02:18
0.078
A*02:280.010
A*02:05
0.005
A*02:13
0.005
A*02:03
0.063
A*02:10
0.386
A*03:01
0.396
A*03:02
0.094
A*11:01
9.117
A*11:02
0.219
A*11:05
0.005
A*24:02 36.475
A*24:20
0.709
A*24:08
0.042
A*24:07
0.021
A*24:04
0.016
A*24:250.010
A*24:05
0.005
A*24:03
0.005
A*26:01
7.350
A*26:03
2.288
A*26:02
1.809
A*26:05
0.068
A*26:06
0.010
A*29:010.010
A*30:01
0.209
A*30:04
0.021
A*31:01
8.403
A*32:01
0.021
A*33:03
7.507
A*33:010.010
A*34:010.010
A*66:01
0.005
A*68:010.010
A*02:53N 0.010
A*24:46
0.010
A*11:43
0.005
A*24:88
0.005
A*26:18
0.005
A*31:11
0.005

Cw1
Cw2
Cw4
Cw5
Cw6
Cw7

Cw8

Cw9
Cw10
Cw12
Cw14
Cw15

Cw17
undeﬁned

HLA-C
alleleHLA-B serological
GF (%)
speciﬁcity

HLA-B
alleleHLA-DRB1
serological
GF (%) speciﬁcity

C*01:02
C*01:03
C*02:02
C*04:01
C*05:01
C*06:02
C*07:02
C*07:04
C*07:01
C*08:01
C*08:03
C*08:02
C*03:03
C*03:04
C*03:02
C*12:02
C*12:03
C*14:02
C*14:03
C*15:02
C*15:05
C*15:10
C*17:01
C*03:23
C*03:43
C*07:15
C*03:64
C*04:15
C*16:0417.604
0.318
0.026
4.410
0.381
0.808
12.714
0.928
0.073
7.355
1.361
0.026
13.05312.3910.568
11.182
0.083
6.871
6.688
3.081
0.016
0.005
0.010
0.010
0.010
0.010
0.005
0.005
0.005

B*07:02
B*07:05
B*07:31
B*08:01
B*13:01
B*13:02
B*18:01
B*56:03
B*55:12
B*27:04
B*27:05
B*27:06
B*35:01
B*35:05
B*35:04
B*35:35
B*37:01
B*38:02
B*38:01
B*39:04
B*39:23
B*39:05
B*39:01
B*39:02
B*41:02
B*44:03
B*44:02
B*45:01
B*46:01
B*46:02
B*48:01
B*50:01
B*51:01
B*51:02
B*51:03
B*52:01
B*53:01
B*54:01
B*55:02
B*55:04
B*56:01
B*57:01
B*58:01
B*59:01
B*40:01
B*40:07
B*40:52
B*40:02
B*40:06
B*40:03
B*40:50
B*40:53
B*40:11

5.630
0.026
0.005
0.016
1.173
0.287
0.005
0.177
0.005
0.188
0.063
0.005
8.263
0.016
0.005
0.005
0.495
0.281
0.016
0.209
0.031
0.005
3.321
0.308
0.005
6.751
0.375
0.005
4.765
0.005
2.878
0.016
8.925
0.235
0.005
11.093
0.005
7.491
2.648
0.130
0.850
0.005
0.579
2.043
5.348
0.010
0.005
7.945
4.791
0.407
0.016
0.005
0.005

B7

B8
B13
B18
B22
B27

B35

B37
B38
B39

B3901
B3902
B41
B44
B45
B46
B48
B50
B51
B5102
B5103
B52
B53
B54
B55
B56
B57
B58
B59
B60

B61

254

DR1
DR4

DR6
DR7
DR8

DR9
DR10
DR11

DR12
DR13

DR14

DR1403
DR1404
DR15

DR16
DR17
undeﬁned

HLADRB1
allele

GF (%)

DRB1*01:01
DRB1*01:02
DRB1*04:05
DRB1*04:06
DRB1*04:03
DRB1*04:10
DRB1*04:01
DRB1*04:07
DRB1*04:04
DRB1*04:02
DRB1*04:11
DRB1*14:45
DRB1*07:01
DRB1*08:03
DRB1*08:02
DRB1*08:09
DRB1*09:01
DRB1*10:01
DRB1*11:01
DRB1*11:06
DRB1*11:08
DRB1*12:01
DRB1*12:02
DRB1*13:02
DRB1*13:01
DRB1*13:07
DRB1*14:54
DRB1*14:05
DRB1*14:06
DRB1*14:07
DRB1*14:02
DRB1*14:12
DRB1*14:29
DRB1*14:03
DRB1*14:04
DRB1*15:02
DRB1*15:01
DRB1*15:04
DRB1*16:02
DRB1*03:01
DRB1*04:57
DRB1*08:23

5.823
0.016
13.491
3.388
3.055
2.153
0.954
0.573
0.255
0.005
0.005
0.005
0.339
8.320
4.207
0.042
14.283
0.474
2.518
0.016
0.005
3.774
1.814
5.948
0.589
0.005
3.274
2.090
1.303
0.109
0.057
0.026
0.016
1.569
0.010
10.650
7.778
0.005
0.897
0.146
0.005
0.005

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tissue Antigens, 2015, 85, 252–259

N. Ikeda et al.

HLA allele and haplotype frequency in Japanese population

Table 2 Continued
HLA-A
serological
speciﬁcity

HLA-A
allele

GF (%)HLA-C serological
speciﬁcity

HLA-C
allele

GF (%)HLA-B serological
speciﬁcity

HLA-B
allele

GF (%)

B62

B*15:01
B*15:07
B*15:27
B*15:28
B*15:35
B*15:25
B*15:38
B*14:01
B*14:02
B*67:01
B*15:18
B*15:11
B*15:02
B*15:13
B*78:02
B*81:01
B*54:21b
B*15:26N
B*35:64
B*51:36
B*52:05
B*52:11

7.585
0.652
0.109
0.016
0.016
0.010
0.005
0.016
0.010
1.225
1.522
0.881
0.031
0.005
0.005
0.005
0.005
0.005
0.005
0.005
0.005
0.005

B64
B65
B67
B71
B75
B77
B78
B81
Unknownb
Null
undeﬁned

HLA-DRB1
serological
speciﬁcity

HLADRB1
allele

GF (%)

GF, gene frequencies; HLA, human leucocyte antigen.
a GF are calculated by using 19,183 haplotypes counted manually. Nomenclature of serological speciﬁcities, refer to Tissue Antigens 2010: 75: 291–455(15).
b
The allele is not in the paper (15).

Statistical analysis

The haplotypes were counted manually using Microsoft Excel®
spreadsheets. The haplotypes that extended three or moregenerations were counted once (Figure 1). The allele andhaplotype frequencies were calculated by using 19,183 haplotypes, counted as mentioned above. Relative LD values (RD)were computed for each haplotypes (17, 18). The exact testfor deviation from Hardy–Weinberg Equilibrium were evaluated by GENEPOP software, version 4.2 (19, 20), which uses
a Markov Chain (MC) algorithm (dememorization = 10,000,
batches = 10,000, and iterations per batch = 10,000) to estimate
the P-value.
The expected prevalence (P) of the allele or the haplotype
under Hardy–Weinberg proportions were calculated from AF
by using the following equation: P = 1 − (1 − AF)2 .

in Table 2 need specific attention for HLA allele matching in
unrelated HSCT between the Japanese, as they are present athigh frequencies within a serotype (allele family) match.
Haplotype segregation analysis based on family study

Haplotype frequencies of HLA-A-C-B-DRB1

Table 3 lists 60 haplotypes with frequencies higher than 0.2%
in the population. Approximately, 38% of the entire Japanese
population is expected to carry one or two of the five mostcommon haplotypes.
These data have also been submitted to Allele Frequency
Net Database (AFND) (22). The four-loci haplotypes withfrequencies equal to or more than 0.01% and the AF can befound at the AFND website, www.allelefrequencies.net (22).

Results

HLA-A-B-DRB1 haplotype sets with the same serotypes

HLA-A, -B, -C, and -DRB1 AF

Table 4 lists the sets of three-loci haplotypes at frequencies of
0.2% or greater, which would have the same serotype.

Table 2 presents the list of the AF of HLA-A, -B, -C, and -DRB1
loci (21). We identified 44 HLA-A, 75 HLA-B, 29 HLA-C,
and 42 HLA-DRB1 alleles and found A*24:02 to be 36.48%,
the highest in Japanese population; thus, it is distributed inapproximately 60% of the population. The alleles underlined© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tissue Antigens, 2015, 85, 252–259

Haplotype frequencies of HLA-C-B

Table 5 lists 64 haplotypes with frequencies higher than 0.1% in the population. Half of these haplotypes have RD values255

N. Ikeda et al.

HLA allele and haplotype frequency in Japanese population

Table 3 HLA–A–C–B–DRB1 haplotype frequencies in Japanesea
A–C–B–DRB1

HF (%)RD

A–C–B–DRB1

HF (%)

RD

8.377
4.473
3.722
2.539
1.866
1.345
1.058
1.001
0.745
0.709
0.652
0.579
0.547
0.542
0.532
0.532
0.464
0.433
0.427
0.422
0.391
0.391
0.386
0.370
0.360
0.344
0.339
0.323
0.323
0.318

0.79
0.75
0.66
0.33
0.54
0.40
0.51
0.13
0.10
0.14
0.09
0.14
0.08
0.30
0.45
0.11
0.10
0.13
0.10
0.05
0.03
0.25
0.12
0.04
0.05
0.15
0.38
0.16
0.50
0.34

A*02:07–C*01:02–B*46:01–DRB1*09:01
A*33:03–C*03:02–B*58:01–DRB1*13:02
A*26:01–C*03:03–B*35:01–DRB1*04:10
A*02:01–C*03:03–B*15:11–DRB1*09:01
A*24:02–C*04:01–B*15:01–DRB1*04:06
A*24:02–C*14:02–B*51:01–DRB1*14:03
A*24:02–C*03:04–B*40:01–DRB1*04:05
A*26:02–C*03:03–B*15:01–DRB1*14:06
A*24:02–C*03:04–B*40:02–DRB1*04:05
A*02:01–C*07:02–B*07:02–DRB1*01:01
A*02:06–C*07:02–B*07:02–DRB1*01:01
A*11:01–C*01:02–B*54:01–DRB1*08:03
A*11:01–C*01:02–B*55:02–DRB1*04:05
A*31:01–C*14:02–B*51:01–DRB1*04:05
A*02:01–C*15:02–B*51:01–DRB1*15:01
A*03:01–C*05:01–B*44:02–DRB1*13:01
A*11:01–C*07:02–B*67:01–DRB1*15:01
A*24:02–C*14:03–B*44:03–DRB1*13:02
A*01:01–C*06:02–B*37:01–DRB1*10:01
A*24:02–C*03:03–B*35:01–DRB1*15:01
A*24:02–C*03:04–B*40:01–DRB1*11:01
A*31:01–C*14:02–B*51:01–DRB1*09:01
A*26:03–C*03:03–B*15:01–DRB1*09:01
A*02:06–C*01:02–B*54:01–DRB1*04:05
A*02:06–C*03:03–B*35:01–DRB1*09:01
A*02:01–C*01:02–B*46:01–DRB1*08:03
A*02:01–C*08:01–B*40:06–DRB1*09:01
A*02:06–C*08:01–B*48:01–DRB1*04:07
A*31:01–C*04:01–B*56:01–DRB1*09:01
A*31:01–C*07:02–B*07:02–DRB1*01:01

0.308
0.308
0.302
0.292
0.292
0.276
0.266
0.261
0.255
0.250
0.250
0.240
0.240
0.240
0.235
0.235
0.235
0.235
0.229
0.229
0.229
0.229
0.224
0.219
0.219
0.214
0.214
0.214
0.214
0.209

0.09
0.54
0.14
0.33
0.09
0.17
0.04
0.20
0.03
0.04
0.04
0.03
0.09
0.03
0.08
0.63
0.19
0.04
0.57
0.03
0.09
0.03
0.10
0.03
0.02
0.04
0.04
0.37
0.25
0.04

A*24:02–C*12:02–B*52:01–DRB1*15:02
A*33:03–C*14:03–B*44:03–DRB1*13:02
A*24:02–C*07:02–B*07:02–DRB1*01:01
A*24:02–C*01:02–B*54:01–DRB1*04:05
A*02:07–C*01:02–B*46:01–DRB1*08:03
A*11:01–C*04:01–B*15:01–DRB1*04:06
A*24:02–C*01:02–B*59:01–DRB1*04:05
A*11:01–C*01:02–B*54:01–DRB1*04:05
A*26:01–C*03:04–B*40:02–DRB1*09:01
A*24:02–C*08:01–B*40:06–DRB1*09:01
A*24:02–C*14:02–B*51:01–DRB1*09:01
A*31:01–C*14:02–B*51:01–DRB1*08:02
A*33:03–C*14:03–B*44:03–DRB1*08:03
A*26:02–C*08:01–B*40:06–DRB1*09:01
A*02:01–C*03:04–B*13:01–DRB1*12:02
A*24:02–C*01:02–B*46:01–DRB1*08:03
A*02:06–C*08:01–B*40:06–DRB1*09:01
A*11:01–C*07:02–B*39:01–DRB1*08:03
A*26:01–C*03:04–B*40:02–DRB1*08:02
A*02:06–C*03:03–B*35:01–DRB1*15:01
A*24:02–C*12:02–B*52:01–DRB1*09:01
A*31:01–C*14:02–B*51:01–DRB1*14:03
A*02:06–C*07:02–B*39:01–DRB1*15:01
A*24:02–C*03:04–B*40:02–DRB1*09:01
A*02:01–C*01:02–B*54:01–DRB1*04:05
A*26:03–C*03:03–B*15:01–DRB1*15:01
A*11:01–C*07:02–B*67:01–DRB1*16:02
A*02:06–C*01:02–B*59:01–DRB1*04:05
A*24:02–C*03:03–B*15:07–DRB1*04:03
A*24:02–C*07:04–B*15:18–DRB1*04:01

HF, haplotype frequencies; HLA, human leucocyte antigen; RD, relative linkage disequilibrium value.
aFour-locus haplotypes with HF >0.2% are listed.

Table 4 Haplotype sets assigned same serotype of HLA–A–B–DRB1a
Set no.

Haplotype

HF (%)

1

A*02:01-B*07:02-DRB1*01:01
A*02:06-B*07:02-DRB1*01:01
A*02:01-B*46:01-DRB1*08:03
A*02:07-B*46:01-DRB1*08:03
A*02:01-B*54:01-DRB1*04:05
A*02:06-B*54:01-DRB1*04:05
A*02:01-B*40:06-DRB1*09:01
A*02:06-B*40:06-DRB1*09:01
A*24:02-B*40:02-DRB1*09:01
A*24:02-B*40:06-DRB1*09:01
A*24:02-B*15:01-DRB1*04:06
A*24:02-B*15:07-DRB1*04:03
A*24:02-B*35:01-DRB1*04:03
A*24:02-B*35:01-DRB1*04:05
A*26:01-B*40:02-DRB1*09:01
A*26:01-B*40:06-DRB1*09:01
A*26:02-B*40:06-DRB1*09:01

0.26
0.25
0.23
1.87
0.38
0.22
0.29
0.48
0.53
0.95
0.31
0.34
0.23
0.22
0.81
0.22
0.54

2
3
4
5
6
7
8

HF, haplotype frequency; HLA, human leucocyte antigen.
a The analysis objects are HF with more than 0.2%.

256

more than 0.7, suggesting conservation of HLA-C-B linkage.
B*40:02 and B*40:06 which correspond to the same serotype(B61) have high frequencies alleles of B61 and need attention for matching in HSCT. Focusing on these, Table 5 shows
the frequency of C*03:04-B*40:02 as 6.26%, while the frequency of B*40:02 is 7.95% (Table 2); therefore, the frequency of C*03:04-B*40:02 linkage would account for 79% of
B*40:02 alleles. Similarly, linkage of B*40:06 with C*08:01
would account for 81% of the presence of B*40:06. Thus,
it is important to differentiate serotypes such as B61 when
the constituting alleles are in linkage with different HLA-C
alleles.
Observed recombination

A total observation number of recombination events were 136
in 134 families. These were divided into two groups: (i) the
haplotypes of 103 parents (75.7%) could be determined, (ii)the haplotypes of 33 parents (24.3%) could not be determined
and thus were inferred. Table 6 summarizes the observation
number of recombination events in informative families whichcontain the parents and three or more children. The genotypes© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tissue Antigens, 2015, 85, 252–259

N. Ikeda et al.

HLA allele and haplotype frequency in Japanese population

Table 5 HLA–C–B haplotype frequencies in Japanesea
C–B

HF (%)RD

C–B

HF (%)

RD

C*12:02–B*52:01
C*01:02–B*54:01
C*14:02–B*51:01
C*14:03–B*44:03
C*03:04–B*40:02
C*03:03–B*35:01
C*07:02–B*07:02
C*01:02–B*46:01
C*08:01–B*40:06
C*07:02–B*39:01
C*03:03–B*15:01
C*03:04–B*40:01
C*04:01–B*15:01
C*01:02–B*55:02
C*01:02–B*59:01
C*08:01–B*48:01
C*15:02–B*51:01
C*07:02–B*67:01
C*03:04–B*13:01
C*08:01–B*35:01
C*03:03–B*40:02
C*07:02–B*40:01
C*08:03–B*48:01
C*07:04–B*15:18
C*03:03–B*15:11
C*15:02–B*40:01
C*01:02–B*15:01
C*03:03–B*15:07
C*03:02–B*58:01
C*04:01–B*56:01
C*07:02–B*15:01
C*06:02–B*37:01

10.963
7.147
6.761
6.641
6.256
6.115
5.573
4.316
3.904
3.196
3.190
2.867
2.606
2.252
2.012
1.553
1.345
1.220
1.069
1.069
1.048
0.923
0.923
0.907
0.850
0.704
0.626
0.610
0.568
0.537
0.500
0.495

0.99
0.94
0.98
0.99
0.76
0.70
0.99
0.89
0.80
0.96
0.33
0.47
0.56
0.82
0.98
0.50
0.38
1.00
0.90
0.07
0.00
0.05
0.67
0.98
0.96
0.18
−0.53
0.93
1.00
0.61
−0.48
1.00

C*08:01–B*15:18
C*03:04–B*51:01
C*04:01–B*35:01
C*04:01–B*40:01
C*03:04–B*40:03
C*05:01–B*44:02
C*03:04–B*35:01
C*03:04–B*40:06
C*15:02–B*40:06
C*01:03–B*46:01
C*07:02–B*39:02
C*15:02–B*40:02
C*06:02–B*13:02
C*07:02–B*38:02
C*03:04–B*15:01
C*03:03–B*55:02
C*03:03–B*48:01
C*01:02–B*56:01
C*03:03–B*40:01
C*08:03–B*54:01
C*01:02–B*51:01
C*07:02–B*39:04
C*01:02–B*56:03
C*15:02–B*51:02
C*01:02–B*40:02
C*12:02–B*27:04
C*15:02–B*15:01
C*08:03–B*15:01
C*03:03–B*55:04
C*07:02–B*40:02
C*04:01–B*15:27
C*04:01–B*48:01

0.495
0.469
0.464
0.464
0.396
0.370
0.339
0.339
0.334
0.318
0.308
0.292
0.287
0.276
0.266
0.261
0.240
0.235
0.235
0.229
0.224
0.209
0.177
0.151
0.141
0.141
0.141
0.136
0.120
0.115
0.109
0.109

0.27
−0.58
0.02
0.05
0.97
0.99
−0.67
−0.43
0.06
1.00
1.00
0.02
1.00
0.98
−0.72
−0.25
−0.36
0.12
−0.66
0.10
−0.86
1.00
1.00
0.63
−0.90
0.72
−0.40
0.03
0.91
−0.89
1.00
−0.14

HF, haplotype frequencies; HLA, human leucocyte antigen; RD, relative linkage disequilibrium value.
aC-B haplotypes with HF >0.1% are listed.
Table 6 Number of HLA–A/C, C/B, B/DRB1 recombination evens by eachfamily structure

Parents

Total

Number ofchildren

Number offamilies

A/Can (%R/T)

B/DRB1an (%R/T)

A/DRB1an (%R/T)

5
4
3
836

5
28
233
266

b

b

b

2 (0.89)
7 (0.50)
9 (0.54)

1 (0.45)
8 (0.57)
9 (0.54)

3 (1.34)
15 (1.07)
18 (1.08)

HLA, human leucocyte antigen.
a X/Y, number of recombination between X and Y; %R/T, % of recombination per transmission.
b
No observation of recombination.

of these parents are heterozygote at all loci of HLA-A, -B, -C,
and -DRB1. Group (ii) is not included in these informative
families. The transmission of recombination (%R/T) showsthe HLA-A-DRB1 recombination probability as 1.08% perchild. Furthermore, Table 6 also indicates the recombination
probabilities of HLA-A-C and B-DRB1 are 0.54%.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tissue Antigens, 2015, 85, 252–259

Comparison to the result by MLE

Table 7 shows the haplotype frequencies of HLA-A-C-B-DRB1
based on family study (result-FS) and based on MLE(result-MLE) with frequencies more than 0.5%. In the frequent
haplotypes with frequencies not less than 0.12%, result-MLE
tends to be higher than result-FS. In the low-frequent
haplotypes with less than 0.12%, result-MLE tends to belower than result-FS. In addition, result-MLE could not be
detected in 585 haplotypes of the 2099 haplotypes withfrequencies less than 0.12% in result-FS.
On the allele data used for this comparison, four loci showed
Hardy–Weinberg Equilibrium: the P-values of the exact test at
HLA-A, -B, -C, and DRB1 loci were 0.3184, 0.2557, 0.1449,
and 0.4998, respectively.
Discussion

The AF presented in Table 2 show the high frequency ofA*24:02 at 36.48%; therefore, around 60% of the population
could be administered peptide vaccines such as WT1 peptide
257

N. Ikeda et al.

HLA allele and haplotype frequency in Japanese population

Table 7 Comparison to maximum likelihood estimation (n = 4500)a
A–C–B–DRB1

FS (%)MLE (%)

A*24:02-C*12:02-B*52:01-DRB1*15:02
A*33:03-C*14:03-B*44:03-DRB1*13:02
A*24:02-C*07:02-B*07:02-DRB1*01:01
A*24:02-C*01:02-B*54:01-DRB1*04:05
A*02:07-C*01:02-B*46:01-DRB1*08:03
A*11:01-C*04:01-B*15:01-DRB1*04:06
A*24:02-C*01:02-B*59:01-DRB1*04:05
A*11:01-C*01:02-B*54:01-DRB1*04:05
A*26:01-C*03:04-B*40:02-DRB1*09:01
A*24:02-C*08:01-B*40:06-DRB1*09:01
A*24:02-C*14:02-B*51:01-DRB1*09:01
A*31:01-C*14:02-B*51:01-DRB1*08:02
A*33:03-C*14:03-B*44:03-DRB1*08:03
A*26:02-C*08:01-B*40:06-DRB1*09:01
A*24:02-C*01:02-B*46:01-DRB1*08:03
A*02:01-C*03:04-B*13:01-DRB1*12:02

8.144%
4.444%
3.689%
2.344%
2.044%
1.322%
1.100%
0.833%
0.778%
0.733%
0.722%
0.589%
0.578%
0.556%
0.522%
0.511%

8.298%
4.478%
3.857%
2.490%
2.053%
1.326%
1.167%
0.815%
0.892%
0.830%
0.803%
0.619%
0.589%
0.526%
0.504%
0.502%

FS, haplotype frequencies based on family study; MLE, haplotype frequencies estimated by maximum likelihood estimation.
a Four-locus haplotypes with frequencies >0.5% in result-FS are listed.

vaccine presented by HLA molecules coded A*24:02 (23, 24).
Moreover, around 80% of the population could be administeredthe vaccines (25, 26) presented by HLA molecules coded by
one of three alleles as A*24:02 (36.5%), A*02:01 (11.6%), andA*02:06 (9.1%).
The haplotype frequencies are also characteristic of Japanese
population. The haplotypes of high frequencies are wellconserved. Approximately one third (38%) of Japanese population carry the five most common haplotypes. We believethat this is strongly influenced by the founder effect: theancestors having the common haplotypes migrated to Japan,
thus, their haplotypes have been concentrated. While searchingin the AFND (22), we found that the common haplotypes
are homologous to those residing in the neighboring countries, especially in South Korea (11, 12, 27–31). In Korean
population, the frequent haplotypes are similar to the onesin Japanese population. The three major Japanese haplotypes A*24:02-C*12:02-B*52:01-DRB1*15:02, A*33:03C*14:03-B*44:03-DRB1*13:02, and A*24:02-C*07:02-B*07:
02-DRB1*01:01 have also high frequencies in Korea (1.9%,
4.2%, 2.9%, respectively) (27), suggesting the migration ofsome ancestors though the Korean Peninsula.
The haplotype analysis not only helps in understanding thehistory of human migration but also in matching for unrelateddonor searches for HSCT. In JMDP, the HLA compatibility
with a donor is evaluated by both serotype and genotype.
Furthermore, allele matching is a better evaluation of compatibility compared to the serotype matching; the rejection
and GVHD risk of bone marrow transplant (BMT) have beenfound to be lower with allele-level matching compared to theserotype-level matching (3, 32). Accordingly, focusing on thealleles in every serotype, A2, A26, etc. increases the possibilityof allele mismatch in spite of serotype match (Table 2). As
258

shown in Table 4, HLA-A2, B61, B15, and DR4 especially
increase the allele mismatch risk. However, compared with
South Korean haplotype analysis, South Korea has 15 setsof haplotypes to pay attention to in HSCT matching (27) but
Japan has eight sets (Table 4). It also shows that for most HLA
haplotypes at the serotype-level, HLA haplotype matching isalmost allele-level matching in HSCT between the Japanese.
Although HLA-C locus is not indispensable for registering
information in HSCT, HLA-C allele matching is important(32, 33). Even without information of donor HLA-C allele,
the C-B haplotypes can predict HLA-C allele, although notalways, because of well-conserved C-B linkage (Table 5); theconservation is possible due to short genetic linkage distance.
In other words, HLA-B allele matching increases the possibilityof HLA-C allele matching. Accordingly, analysis in the HLA
distribution in Japanese population may contribute in planning
the strategies of HLA matching for HSCT.
Result-MLE was similar to result-FS (Table 7). Table 7 indicates the haplotype frequencies estimated by the software arevery similar to real family-derived haplotypes. If the detection of the low frequency haplotypes is needed, determinationof haplotypes by descent or a large sample size appears to benecessary.
In conclusion, this study of determination of HLA allele andhaplotype frequencies using family samples not only serves as atool for elucidating linkage of each HLA locus but also acts as atool in detecting HLA gene mutations in human germ cells suchas recombination. The data obtained in this study will be usefulin various fields such as anthropology, transplantation therapy,
and disease association studies.

Acknowledgments

We would like to thank Jun Ohashi from Department of Biological Sciences, Graduate School of Science, The Universityof Tokyo, for valuable comments and suggestions about choosing unrelated samples and the exact test for deviation from
Hardy–Weinberg equilibrium.

Conﬂicts of interest

The authors have no conflicts of interest.

References
1. Cao K, Hollenbach J, Shi X et al. Analysis of the frequencies of
HLA-A, B, and C alleles and haplotypes in the five major ethnic
groups of the United States reveals high levels of diversity inthese loci and contrasting distribution patterns in these
populations. Hum Immunol 2001: 62: 1009–30.
2. Imanishi T, Wakisaka A, Gojobori T. Genetic relationships
among various human populations indicated by MHC
polymorphisms. In: Tsuji K, Aizawa M, Sasazuki T, eds. HLA
1991. Oxford: Oxford University Press, 1992, 627 –32.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tissue Antigens, 2015, 85, 252–259

N. Ikeda et al.

3. Lee SJ, Klein J, Haagenson M et al. High-resolution
donor-recipient HLA matching contributes to the success of
unrelated donor marrow transplantation. Blood 2007a: 110:
4576–83.
4. Horan J, Wang T, Haagenson M et al. Evaluation of HLA
matching in unrelated hematopoietic stem cell transplantation for
nonmalignant disorders. Blood 2012: 120: 2918–24.
5. Morishima S, Ogawa S, Matsubara A et al. Impact of highly
conserved HLA haplotype on acute graft-versus-host disease.
Blood 2010: 115: 4664–70.
6. Nakaoka H, Mitsunaga S, Hosomichi K et al. Detection ofancestry informative HLA alleles confirms the admixed origins
of Japanese population. PLoS One 2013: 8: e60793.
7. Tokunaga K, Ishikawa Y, Ogawa A et al. Sequence-based
association analysis of HLA class I and II alleles in Japanesesupports conservation of common haplotypes. Immunogenetics
1997: 46: 199–205.
8. Saito S, Ota S, Yamada E, Inoko H, Ota M. Allele frequencies
and haplotypic associations defined by allelic DNA typing at
HLA class I and class II loci in the Japanese population. Tissue
Antigens 2000: 56: 522–9.
9. Moriyama Y, Kato K, Mura T, Juji T. Analysis of HLA genefrequencies and HLA haplotype frequencies for bone marrowdonors in Japan. MHC 2006: 12: 183–201.
10. Ogata S, Shi L, Matsushita M et al. Polymorphisms of humanleucocyte antigen genes in Maonan people in China. Tissue
Antigens 2007: 69: 154–60.
11. Yao Y, Shi L, Shi L et al. Distribution of HLA-A, -B, -Cw, and-DRB1 alleles and haplotypes in an isolated Han population inSouthwest China. Tissue Antigens 2009: 73: 561–8.
12. Shi L, Yao YF, Shi L et al. HLA alleles and haplotypes
distribution in Dai population in Yunnan province, Southwest
China. Tissue Antigens 2010: 75: 159–65.
13. Shichi D, Kikkawa EF, Ota M et al. The haplotype block,
NFKBIL1-ATP6V1G2-BAT1-MICB-MICA, within the class III- class I boundary region of the human major histocompatibility
complex may control susceptibility to hepatitis C virus-associateddilated cardiomyopathy. Tissue Antigens 2005: 66: 200–8.
14. Kamatani Y, Wattanapokayakit S, Ochi H et al. A genome-wide
association study identifies variants in the HLA-DP locus
associated with chronic hepatitis B in Asians. Nat Genet 2009:41: 591–5.
15. Zhao H, Qi Y, Wang Y et al. Interactive contribution ofserine/threonine kinase 39 gene multiple polymorphisms tohypertension among northeastern Han Chinese. Sci Rep 2014: 4:5116.
16. Wang ZT, Hu JJ, Fan R, Zhou J, Zhong J. RAGE gene threepolymorphisms with Crohn’s disease susceptibility in Chinese
Han population. World J Gastroenterol 2014: 20: 2397–402.
17. Lewontin RC. The Interaction of Selection and Linkage. I.
General Considerations; Heterotic Models. Genetics 1964: 49:
49–67.
18. Hedrick PW. Gametic disequilibrium measures: proceed withcaution. Genetics 1987: 117: 331–41.
19. Raymond M, Rousset F. GENEPOP (version 1.2): population
genetics software for exact tests and ecumenicism. Heredity
1995: 86: 248–9.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tissue Antigens, 2015, 85, 252–259

HLA allele and haplotype frequency in Japanese population

20. Rousset F. GENEPOP’007: a complete re-implementation of the
GENEPOP software for Windows and Linux. Mol Ecol 2007: 8:
103–6.
21. Marsh SGE, Albert ED, Bodmer WF et al. Nomenclature forfactors of the HLA system, 2010. Tissue Antigens 2010: 75:
291–455.
22. Gonzalez-Galarza FF, Christmas S, Middleton D, Jones AR.
Allele frequency net: A database and online repository for
immune gene frequencies in worldwide populations. Nucleic
Acids Res 2011: 39: D913–9.
23. Tsuboi A, Oka Y, Udaka K et al. Enhanced induction of human
WT1-specific cytotoxic T lymphocytes with a 9-mer WT1
peptide modified at HLA-A*2402-binding residues. Cancer
Immunol Immunother 2002: 51: 614–20.
24. Oka Y, Tsuboi A, Taguchi T et al. Induction of WT1 (Wilms’
tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide
vaccine and the resultant cancer regression. Proc Natl Acad Sci
U S A 2004: 101: 13885–90.
25. Kaida M, Morita-Hoshi Y, Soeda A et al. Phase 1 trial of Wilms
tumor 1 (WT1) peptide vaccine and gemcitabine combination
therapy in patients with advanced pancreatic or biliary tract
cancer. J Immunother 2011: 34: 92–9.
26. Ohno S, Okuyama R, Aruga A, Sugiyama H, Yamamoto M.
Phase I trial of Wilms’ Tumor 1 (WT1) peptide vaccine with
GM-CSF or CpG
in patients with solid malignancy. Anticancer Res 2012: 32:
2263–9.
27. Lee KW, Oh DH, Lee C, Yang SY. Allelic and haplotypic
diversity of HLA-A, -B, -C, -DRB1, and -DQB1 genes in the
Korean population. Tissue Antigens 2005: 65: 437.
28. Yang G, Deng YJ, Hu SN et al. HLA-A, -B, and -DRB1
polymorphism defined by sequence based typing of the Han
population in Northern China. Tissue Antigens 2006: 67:
146–52.
29. Chen S, Hu Q, Xie Y et al. Origin of Tibeto-Burman speakers:
evidence from HLA allele distribution in Lisu and Nu inhabiting
Yunnan of China. Hum Immunol 2007: 68: 550–9.
30. Lai MJ, Wen SH, Lin YH et al. Distributions of human leukocyte
antigen-A, -B, and -DRB1 alleles and haplotypes based on
46,915 Taiwanese donors. Hum Immunol 2010: 71: 777–82.
31. Wen SH, Lai MJ, Yang KL. Human leukocyte antigen-A, -B, and-DRB1 haplotypes of cord blood units in the Tzu Chi Taiwan
Cord Blood Bank. Hum Immunol 2008: 69: 430–6.
32. Flomenberg N, Baxter-Lowe LA, Confer D et al.
Impact of HLA class I and class II high-resolution
matching on outcomes of unrelated donor bone marrowtransplantation: HLA-C mismatching is associated with astrong adverse effect on transplantation outcome. Blood 2004:
104: 1923–30.
33. Kanda Y, Kanda J, Atsuta Y et al. Impact of a single humanleucocyte antigen (HLA) allele mismatch on the outcome ofunrelated bone marrow transplantation over two time periods. A
retrospective analysis of 3003 patients from the HLA Working
Group of the Japan Society for Blood and Marrow
Transplantation. Br J Haematol 2013: 161: 566–77.

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