© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

LETTERSA SNP in the ABCC11 gene is the determinant of humanearwax typeKoh-ichiro Yoshiura1,2, Akira Kinoshita1,2, Takafumi Ishida3, Aya Ninokata3, Toshihisa Ishikawa4,
Tadashi Kaname2,5, Makoto Bannai6, Katsushi Tokunaga6, Shunro Sonoda7, Ryoichi Komaki8, Makoto Ihara9,
Vladimir A Saenko10, Gabit K Alipov11, Ichiro Sekine11, Kazuki Komatsu12, Haruo Takahashi12,
Mitsuko Nakashima1,2,13, Nadiya Sosonkina1,2, Christophe K Mapendano1,2, Mohsen Ghadami1,2,
Masayo Nomura1,2,14, De-Sheng Liang2,15, Nobutomo Miwa1,2, Dae-Kwang Kim16, Ariuntuul Garidkhuu17,
Nagato Natsume17, Tohru Ohta2,18, Hiroaki Tomita19, Akira Kaneko20, Mihoko Kikuchi21,
Graciela Russomando22, Kenji Hirayama21, Minaka Ishibashi23, Aya Takahashi23, Naruya Saitou23,
Jeffery C Murray24, Susumu Saito25, Yusuke Nakamura25,26 & Norio Niikawa1,2
Human earwax consists of wet and dry types. Dry earwax isfrequent in East Asians, whereas wet earwax is common inother populations. Here we show that a SNP, 538G-A(rs17822931), in the ABCC11 gene is responsible fordetermination of earwax type. The AA genotype corresponds to
dry earwax, and GA and GG to wet type. A 27-bp deletion in
ABCC11 exon 29 was also found in a few individuals of Asianancestry. A functional assay demonstrated that cells with allele
A show a lower excretory activity for cGMP than those withallele G. The allele A frequency shows a north-south and eastwest downward geographical gradient; worldwide, it is highest
in Chinese and Koreans, and a common dry-type haplotype isretained among various ethnic populations. These suggest thatthe allele A arose in northeast Asia and thereafter spreadthrough the world. The 538G-A SNP is the first example
of DNA polymorphism determining a visible genetic trait.
Earwax (cerumen) is a secretory product of ceruminous apocrine
glands. Human earwax is a mendelian trait consisting of wet and dry
types1. The wet earwax is brownish and sticky, whereas the dry typelacks cerumen. The wet cerumen phenotype is completely dominant

to the dry type. The dry type is seen frequently (80–95%) among East
Asians2–4, but uncommon (0–3%) in populations of European and
African origins. Intermediate frequencies (30–50%) of the dry type areseen in populations of Southern Asia, the Pacific Islands, Central Asia
and Asia Minor, as well as among the Native North American andInuit of Asian ancestry2,5. These figures show geographical gradient
distributions. Earwax type may play a role in the axillary odor andpossibly in breast cancer susceptibility6, although the association withbreast cancer remains controversial7. Using a linkage analysis, we havepreviously assigned the earwax gene locus to the pericentromericregion of chromosome 16 (ref. 8).
With the aim of further mapping of the earwax locus, we performed
genotyping and case-control study of 64 Japanese individuals with dry
earwax and 54 with the wet type using 134 CA repeat markers.
Although 12 markers showed an association with a P value of log10
o –2.3, they did not allow finer mapping because their distribution
was too wide. Nevertheless, six of them were located to a B2.5-Mb
pericentromeric region of 16q. Another association study using
36 SNPs of the samples showed that two SNPs (B81540.1 and
IMS-JST141676) at this region gave P values of 4.0  10–9 and7.6  10–6, respectively. All 64 individuals with dry earwax were

1Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. 2Core Research for Evolutional Science andTechnology (CREST), Japan Science and Technology, Kawaguchi, Japan. 3Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo,
Japan. 4Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan. 5Department ofMedical Genetics, University of the Ryukyu School of Medicine, Okinawa, Japan. 6Department of Human Genetics, Graduate School of Medicine, University of Tokyo,
Tokyo, Japan. 7Research and Development Center, Kagoshima University, Kagoshima, Japan. 8Cosmetics Laboratory, Kanebo Cosmetics, Inc., Odawara, Japan.
9Department of Radiation Biophysics, 10International Health and Radiation Research, 11Molecular Pathology, 12Otolaryngology, 13Plastic and Reconstructive Surgery
and 14Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. 15National Laboratory of Medical Genetics of China, Xiangya
Hospital, Central South University, Changsha, China. 16Department of Anatomy, Hanvit Institute for Medical Genetics, Keimyung University School of Medicine, Taegu,
Korea. 17Graduate School of Dentistry, Aichi Gakuin University, Nagoya, Japan. 18Research Institute of Personalized Health Sciences, Health Science University of
Hokkaido, Ishikari-Tobetsu, Japan. 19Human Genetics Laboratory, Department of Psychiatry and Human Behavior, University of California, Irvine, USA. 20Malaria
Research Laboratory, Karolinska University Hospital, Stockholm, Sweden. 21Department of Molecular Immunogenetics, Institute of Tropical Medicine, Nagasaki
University, Nagasaki, Japan. 22Departamento de Biologia Molecular, Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asuncion, Paraguay.
23Division of Population Genetics, National Institute of Genetics, Mishima, Japan. 24Department of Pediatrics, University of Iowa, Iowa City, USA. 25Laboratory for SNP
Analysis, SNP Research Center, The Institute of Physical and Chemical Research (RIKEN), Tokyo, Japan. 26Human Genome Center, Institute of Medical Science,
University of Tokyo, Tokyo, Japan. Correspondence should be addressed to K.-i.Y. (kyoshi@net.nagasaki-u.ac.jp).

Received 3 October 2005; accepted 13 December 2005; published online 29 January 2006; doi:10.1038/ng1733

324

VOLUME 38 [ NUMBER 3 [ MARCH 2006 NATURE GENETICS

LETTERS

0100

ABCC12

ABCC11 LONPL SIAH1

200

300

400

500

N4BP1
600

kb
700

–1.0
–2.0
–3.0

B334000
AC007600-M1
B404940–5.0
IMS-JST141676–6.0
P value© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

–4.0

–7.0
–8.0
–9.0

B81540

–10.0
–11.0
–12.0

rs6500380(IVS12+7508A→G)

–13.0
–14.0
–15.0
log10

ss49784070(IVS14+320A→G)

rs17822931(538G→A)
CA repeat marker

SNP marker

Figure 1 Four genes, ABCC12, ABCC11, LONPL and SIAH1, and 31
polymorphic DNA markers located in the B600-kb region between B81540
and IMS-JST141676. Three intragenic polymorphisms, rs17822931(538G-A) in ABCC11, rs6500380 (IVS12+7508A-G) and ss49784070(IVS14+320A-G) in LONPL, gave a P value of o2.0  10–14 in an
association study of 118 volunteers (64 dry-type and 54 wet-type) whose
DNA was collected in anonymous manner and their earwax type selfdeclared, and were in strong linkage disequilibrium (r2 E 1.00) in theJapanese population. Further genotyping in another series of 126 Japaneseindividuals (88 with dry earwax and 38 with wet earwax) whose earwax
types were clinically confirmed showed that rs17822931 is the determinantof earwax type.

homozygotes for one allele each, consistent with an autosomal recessive
trait and supporting the presence of the earwax locus in this region.
According to GenBank, the two-SNP interval is approximately 600 kb
and contains five genes: ABCC12, ABCC11, LONPL, SIAH1 andN4BP1. We performed an association study of the 118 samples using
37 SNPs within the five-gene interval and found that rs17822931(538G-A, G180R) in ABCC11 exon 4, rs6500380 (IVS12+7508A-G)
in LONPL intron 12 and ss49784070 (IVS14+320A-G) within an
Alu-repetitive sequence in LONPL intron 14 showed the lowest
P values (o2.0  10–14; Fig. 1). Genotyping at the three SNP locishowed that 63 of 64 samples from individuals with the dry type were
AA-AA-AA and one GA-GA-GA; of the samples from individuals with
wet earwax, 6 of the 54 were GG-GG-GG, 33 were GA-GA-GA and 15
were AA-AA-AA (Supplementary Table 1 online). We performed LD
analysis for the 37 SNPs and found two LD blocks among the 118
Japanese: one large block containing the 3¢-region of PHKB, the neighboring gene B450 kb centromeric to ABCC12, and another blockincluding the three SNPs as well as SIAH1 (Fig. 2). The three SNPs
were in strong LD (r2 E 1.0) in the Japanese, and only two haplotypes,
G-G-G and A-A-A, were predicted in the Japanese in our samples.
Although these findings suggest that one of the three SNPs defines
earwax type, some samples showed phenotype-genotype inconsistency.

NATURE GENETICS VOLUME 38 [ NUMBER 3 [ MARCH 2006

Genotyping of a new series of 126 Japanese individuals whose
earwax types were identified otologically showed that 87 of 88
individuals with dry earwax were AA homozygotes, and all
38 individuals with the wet type were either GA heterozygotes or
GG homozygotes at each of the three SNP loci (Table 1 and Supplementary Table 1 online). Among the SNPs, only rs17822931 isnonsynonymous (G180R); rs6500380 does not create any splicing
sites nor affect splicing factor binding motifs or known promoter
sequences, and ss49784070 is located within the Alu-repetitive
sequence, all supporting rs17822931 as an earwax determinant. One
exceptional individual showed discordance between phenotype (dryearwax) and genotype (GA heterozygous for each of SNP); sequencing
showed that he had a 27-bp deletion (3939–3965del27, or D27) in
ABCC11 exon 29 (the exon numbering is according to a previous
report9) downstream of the G/A site of the gene. The D27 was
found in his G allele in ABCC11 (Fig. 3). These may support anidea of loss-of-function of the allele G, leading to the dry phenotype.
Thus, the frequency of the dry earwax phenotype among residentsof Nagasaki, Japan is 0.698 (88/126), and that of allele A at rs17822931
is 0.829 (206/252).
We genotyped a total of 33 different populations around the world
at rs17822931. East Asians have high frequencies of allele A. The
highest value (100%) was observed in Northern Han Chinese andKoreans, and the second highest in Mongolians, Chinese from otherareas of China and mainland Japanese. Africans from various
sub-Saharan nations and African Americans showed the lowest
frequency. Intermediate frequencies were observed in other populations (Table 1 and Supplementary Table 2 online). A world mapof the frequency of allele A shows a north-south downward
gradient from Northern China toward Japan and Southern Asia as
well as an east-west gradient from Siberia toward Europe (Fig. 4),
similar to that found previously for earwax phenotype2–5. We also
genotyped for D27 in various populations. Notably, it was foundin 2 of 20 native North Americans, 1 of 10 Andeans, 9 of 30 Bolivians
and 1 of 50 Thai individuals, in addition to 1 of 334 Japaneseindividuals, but none in other populations. All 14 individuals
were heterozygous for D27. As D27 seems independent of genotype
at rs17822931, it is a deletion polymorphism that is relatively
frequent in subsets of the Native Americans. However, as the deletion
among Bolivians was located in an LD block next to the block
involving rs17822931 (Fig. 2c), it remains to be investigatedwhether D27 has a single origin. Analysis of an rs17822931rs6500380-ss49784070 haplotype structure showed a haplotype
G-G-G in all ten African-Americans and in Japanese with wet
earwax, suggesting that it is the ancestral state. In Chinese individuals,
Native Americans, Bolivians and the CEPH family members, thecalculated r2 value between rs6500380 and ss49784070 was 1.00,
whereas the values between rs17822931 and rs6500380 in these
populations and Southern Chinese were 0.82, 0.58, 0.62 and 0.67,
respectively. In all populations examined but the CEPH families,
A-A-A was the major haplotype with variable frequencies(0.47–0.90), whereas G-G-G was the major haplotype in the CEPH
families (Supplementary Table 3 online). We constructed LD blocks,
which showed almost the same structure among the Japanese,
Native Americans, Bolivians and Chinese (Fig. 2). Several recombinations between rs17822931 and rs6500380 were evident inall ethnic groups in which haplotypes were analyzed. Two other
SNPs (B334000 and B404940) gave P values of 1.3  10–3 and1.5  10–3 in an association study of 87 Japanese individuals withdry earwax for respective minor alleles. In addition, a CA repeat
marker (AC007600M1) in intron 21 of ABCC12 (Fig. 1) did not show325

LETTERS

18

19

52

9

23

73

73
60

14

18

79

29

N4BP1

4

Block 2 (159 kb)

9Block 3 (75 kb)

12

23

24

8

47

45
45

76
72

61

29

78
49

B-493890

B-499900

B-501360

B-521140

15

16

17

18

20

21

49

14

76
29

66
6

58

46

7

11

3

26

23

20

37

16
15

5
25
67

56

38
6

10

26

71
14

20

16

28

24

28

60

38

9

8
8

1
13

18
14

40
46

31
22

27

42
52
18

10

41

1

62

27

76
10

60

38

1
9

4

41

73
62

24

47

9

45
16

16
45

26

42
36

9

9
30

42

36

47
9

41
59

19

24

15
36

55
41

11

19
16

70

11

0
5

0

41

4

78

4

12

38

63
32

14

5

7

37
37

9

23
15

50

46

89
61

49
16

25

Block 2 (18 kb)

22

22
8

41

8

13

23

25
57

5

10

65
65

91

12

7

10

75
65

3
81

82

11
23

42
6

63

85

9

8
8

77

B-631128

B-482500

14

Block 1 (142 kb)

1

B-612860

B-475340

LONPL

B-414730

5B-404940

4B-347380

28

B-334000

26

B-333740

25

B-158110

22

B81540-2

B565600

19

1
Block 1 (260 kb)

2
2

SIAH1

ABCC12

B-41250

B-50000017E-locusB-493890

16

IMS-JST
141676

B-482500

15

B-631128

B-475340

13

B-612860

B-404940

B-357760∆27
7B-334000

B-158110

B81540-2

5

2

11

0

0

ABCC11d

N4BP1

B-357760

SIAH1
LONPL

IMS-JST
095062

ABCC1133

079

ABCC12

0
0

0

23
33

33

51

52

39
45

13
21

8
8

52

9

0

0

44
44

29

12

c

44

15

24
50

E-locus

43

36

71
39

84

45

B-41250© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

9

84

52

50
44

77

43
45

68

70

63
66

28

52

20

68
28

86

26

24

24

B565600

025

Block 2 (18 kb)

22
20

39

E-locus17

IMS-JST
141676

16

IMS-JST
141676

12

IMS-JST
095062

9

IMS-JST
095062B-500000

15

Block 1 (159 kb)

B-631128

B-499900

13

N4BP1

B-612860

B-493890

8B-482500

7B-475340

5

SIAH1
LONPL

B-404940

4B-357760∆27

B-333740

1

B-334000

B-158110

ABCC12

B565600

ABCC11b

N4BP1

B81540-2

ABCC11SIAH1
ABCC12LONPL

PHKB

B-41250

a

33
45

12
38

39
2

40
47

Figure 2 Linkage disequilibrium (LD) analysis of candidate region. Three LD blocks were identified in the Japanese. (a) One block at the most centromericregion includes the PHKB gene, another at the middle contains ABCC12, ABCC11, LONPL and SIAH1 and the other at the most telomeric region includes
N4BP1. A haplotype block including rs17822931, rs6500380 and ss49784070 was observed in Native Americans (b), Native Bolivians (c) andSouthern Han Chinese (d). Because of few informative SNPs in the Chinese, we could not define any clear LD blocks other than the block constructed by
the three SNP sites. Also, we did not observe any LD blocks in African-American by the SNPs we used. SNPs used for haplotype analyses are listed in
Supplementary Table 4.

homozygosity among Japanese individuals with dry earwax. Thus,
some recombinations must have occurred even in the Japanese,
although we did not detect any recombination among rs17822931,
rs6500380 and ss49784070. Thus, the high frequency of the dryearwax haplotype A-A-A with little recombination suggests that it has
risen rapidly in frequency from a recent founder. Rare mutation(s) atother site(s) such as D27 in ABCC11 also contribute to dry earwax
formation in a subset of individuals.
We compared the cGMP transport activity of two LLC-PK1 celllines expressing human ABCC11, one carrying allele G (LLC-PK1-G)
and another carrying allele A (LLC-PK1-A). ABCC11 encodes themultidrug resistance–associated protein 8 (MRP8) that consists of
1,382 amino acids and contains two ATP-binding domains and 12transmembrane domains9–11. LLC-PK1-A cells expressed ABCC11mRNA three times more abundantly than LLC-PK1-G (Fig. 5a).
ATP-dependent cGMP transport activity of plasma membrane vesicles
prepared from LLC-PK1-G cells expressing the Gly180 type of MRP8
was significantly higher than the activity of cells expressing the Arg180
type, as measured in cGMP concentrations ranging from 100 mM to
500 mM (Fig. 5b). On the other hand, transport activity of the326

membrane expressing MRP8 with Arg180 was similar to that of mock
vector-transfected LLC-PK1 cells.
The G180R substitution in rs17822931 (538G-A) is located at thefirst transmembrane domain of MRP8. In addition, D27 in exon 29
was found on allele G in a G/A-heterozygous individual with dry
earwax. These may lead to loss of function of the protein andindeed strengthen the evidence that ABCC11 has a role in determining
the human earwax trait. A recent study demonstrated that MRP8
is an amphipathic anion transporter and functions as a drug efflux
pump for purine and pyrimidine nucleotide analogs such as cAMPand cGMP12. Because MRP8 can transport a variety of lipophiles13,
and earwax contains various aliphatic compounds, aromatic hydrocarbons and other molecules14, it is reasonable that wild-type MRP8
functions to secrete earwax. Histological findings of the wet-type
ceruminous gland containing a ‘light granule’ that might be asecretory vesicle15 also support the role of ABCC11. Individuals withfatal surfactant deficiency (that is, a genetic disorder caused bymutations in ABCA3, one of the ABC transporter genes) lacksurfactant in their alveolar type II cells16. Similarly, as no light granules
and microvilli on the cell surface are observed in the dry-type

VOLUME 38 [ NUMBER 3 [ MARCH 2006 NATURE GENETICS

LETTERS
Table 1 Genotypes at the rs17822931 site and frequencies of alleles A and D27 of ABCC11 among different ethnic populations
No. of individuals with genotypes
No. ofEthnic© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

populationsindividuals Frequency
GA

Frequency

GG genotyped of allele A of allele D27

Collected

Tribes or inhabitants studiedAA(frequency)

KoreanTaegu inhabitant

99(1.000)

0

0

99

1.000

ChineseShanxi inhabitant (Northern Han Chinese)74(1.000)

0

0

74

1.000

N.S.

Jingsu inhabitant (Northern Han Chinese)

110(0.924)

9

0

119

0.962

N.S.

Inhabitants of Fujian, Guanjgdong and Hunan Provinces

249(0.750)

63

8

332

0.845

N.S.

0/190

by
D.-K.K.

(Southern Han Chinese)
Han Chinese from multi-regions

42(0.808)

10

0

52

0.904

0/104

D.S.L.

Mongolian

Khalkha tribe

126(0.759)

36

4

166

0.867

0/252

T.I., A.G.

Japanese

Nagasaki inhabitant (western prefecture of Japan mainland)

87(0.690)

35a

4

126

0.829

1/668

N.N.

Okinawa people (southwestern prefecture of Japan)

30(0.517)

25

3

58

0.733

Yonakuni islander (western island of Japan)

13(0.433)

15

2

30

0.683

0/60

S.S.

0/116

T.I., N.N.

T.K.

Ainu in Biratori-Nibutani village in Hokkaido

32(0.552)

24

2

58

0.759

Vietnamese

People from multi-regions

82(0.536)

60

11

153

0.732

Dravidian

Inhabitants of Tamil Nadu in India

27(0.540)

17

6

50

0.710

0/100

T.I.

Northern Thai (Lahu, Shan, Lisu, Hmong, Akha, Mlabri and 215(0.505)

163

48

426

0.696

0/158

T.I., K.H.

(0.633)

10

8

49

0.735

Thai

Karen (Mae-sot Thai) tribes combined)
Central Thai in Bangkok

N.Na.

(Karen)
31

1/100

T.I.

(Bangkokian)Southern Thai (Urak Lawoi and Sakai tribes combined)2(0.026)

23

52

77

0.175

Vedda

Native people in Sri Lanka

7(0.350)

12

1

20

0.650

IndonesianDayak tribe in Kalimantan

12(0.293)

23

6

41

0.573

T.I.

Toraja and Bugis tribes in Sulawesi

27(0.270)

49

24

100

0.515

T.I.
T.I.

MalaysianTaiwanese

T.I.

Floresian18(0.300)

25

17

60

0.508

9

(0.180)

16

25

50

0.340

T.I.

Dani tribe in Irian Jaya

0(0.000)

2

31

33

0.030

T.I.

Sabah in North Borneo

24(0.393)

27

10

61

0.566

0/132 (Sabah)

K.H.

Bentong tribe

8(0.113)

40

23

71

0.394

0/138

K.H.

Taiwan Aborigine (Yami and Ami combined)

34(0.330)

48

21

103

0.563

0/100 (Ami)

T.I.

6

(0.300)

8

6

20

0.500

2/40

R.K.

PalawanEaster Islander
Bolivian

T.I.

Sumbanese

Native American
Philippino

0/52 (Sakai)

Aymara inhabitants

11(0.229)

23

14

48

0.469

T.I.

4

(0.138)

18

7

29

0.448

S.S.

5

(0.167)

14

11

30

0.400

6

(0.200)

11

13

30

0.383

Ayoreos

2(0.040)

34

14

50

0.380

0/98

Sanapana

0(0.000)

14

61

75

0.093

0/150

K.H.

Russian

5(0.045)

45

62

112

0.246

0/208

V.A.S.

Solomon Islander

2(0.323)

25

35

62

0.234

0/122

K.H.

Pacific islander

1(0.143)

1

5

7

0.214

0/14

Coriell

KazakhNative Paraguayan

FrenchFrom the CEPH families

Andean people
HungarianJewishAshkenazi

Ukrainian

9/60

S.S.
G.K.A.
K.H.

1(0.083)

3

8

12

0.208

0/24

CEPH

1

(0.100)

2

7

10

0.200

1/20

Coriell0(0.000)

4

6

10

0.200

0/20

Coriell0(0.000)

4

6

10

0.200

0/20

Coriell
V.A.S.

0(0.000)

15

27

42

0.179

0/84

Papua, New Guinea

1(0.026)

11

26

38

0.171

0/68

T.I.

European American

From CEPH families without the French and Venezuelans

1(0.012)

16

65

82

0.110

0/164

CEPH

Vanuatu islander

Aneityum and Santo islanders combined

1(0.011)

17

74

92

0.103

0/266

K.H.

Papuan(Any and Gau
islanders)
Iberian0(0.000)

2

8

10

0.100

Colombian0(0.000)

2

15

17

0.059

0/34

S.S.

Inhabitants of Ye’Kuana and Sanuma villages

0(0.000)

3

29

32

0.047

0/64

S.S.,CEPH

From various sub-Saharan nations

0(0.000)

1

10

11

0.045

0/22

C.K.M.

0(0.000)

0

10

10

0.000

0/20

Coriell

Venezuelan
AfricanAfrican American

Coriell

D.-K.K., D.-K. Kim; N.S., N. Saitou; N.N., N. Niikawa; D.-S.L., D.-S. Liang; T.I., T. Ishida; A.G., A. Garidkhuu; T.K., T. Kaname; S.S., S. Sonoda; N.Na., N. Natsume; K.H., K. Hirayama; R.K.,
R. Komaki; G.K.A., G.K. Alipov; V.A.S., V.A. Saenko; C.K.M., C.K. Mapendanno; Coriell, from the Coriell Institute; CEPH, from the CEPH families.
aOne exceptional case of dry cerumen who has D27 on the G allele is included.

NATURE GENETICS VOLUME 38 [ NUMBER 3 [ MARCH 2006

327

LETTERSab
rs17822931
Exon 29

G G
W ∆27

A A
#

W W

2/20

1/334

*

GACATGGAGACAGA CACCCTGATCCAGCGCACA
AGCCTTCCAGGGCTGCACC

Figure 3 Pedigree analysis of an individual showing phenotype-genotype
discrepancy (at rs17822931). (a) Family tree and genotypes at
rs17822931 and exon 29 in ABCC11. Filled symbols: wet earwax; open
symbols, dry earwax. Family analysis clearly indicates that allele D27 foundin the proband with dry cerumen (arrow) was transmitted from his father andshows that alleles G and D27 are on the same chromosome and compose
a haplotype. (b) Electropherogram of exon 29 from the proband. A 27-bp
intraexonic deletion is evident in the proband.

ceruminal gland15, earwax may be relevant to the surfactant production and secretion process.
It is believed that ancient Northeast Eurasians had reached a highfrequency of dry earwax, and the population thereafter expanded insize, showing geographic range among East Asians2. Although ourdata showing gradient distributions of allele A with a peak in North
China and Korea favor that hypothesis, its origin should be verified bymutational diversity analysis on the haplotype carrying allele A. An
independent origin of the same mutation (538G-A) is unlikely,
because Southern Chinese individuals, Japanese individuals, Native
Americans, the CEPH families and Native Bolivians with dry earwax
all share the haplotype carrying allele A. Therefore, the geographicalcline of dry earwax may reflect migration of ancient Northeast
Eurasians. This expansion of the dry type may be a result of a certain
selective advantage of the dry cerumen2. The genomic region containing four genes, ABCC12, ABCC11, LONPL and SIAH1, is indeed a
SNP desert that may reflect such strong directional selection important for the modern human evolution17. Furthermore, according to
the International HapMap Project, rs17822931 is one of 32 highdifferentiation, nonsynonymous SNPs that may have experienced
geographically restricted selection pressures18. The high frequency ofallele A in ABCC11 among Native Americans may reflect theirancestors’ migratory wave(s) from Siberia over the Bering land bridgeto the American continent19–21. In this context, D27 found in subsets
of Native North and South Americans, in addition to one subset eachof Japanese and Thai individuals, is of great interest. However, where
and when it originated remains unknown.
The phenotypic implications of earwax remain unknown. Insect
trapping, self-cleaning and prevention of dryness of the external

Figure 5 ABCC11 expression and cGMP transport in LLC-PK1 cells
transfected with allele A and those with allele G. (a) Expression of ABCC11mRNA was compared with GAPDH expression from same cells, using
quantitative PCR. Results from three independent experiments are shown(mean ± s.e.m.). LLC-PK1-A cells expressed ABCC11 three times moreabundantly (3.24 ± 0.64) than LLC-PK1-G (*P o 0.001, Student’s t-test).
(b) ATP-dependent transport of cGMP in plasma membrane vesicles
prepared from ABCC11-expressing LLC-PK1 cells stably expressing Arg180
and Gly180 types of MRP8 and from mock-transfected LLC PK1 cells(mean ± s.d.). Km values calculated for cGMP were 7.8 mM and 302 mM,
representing MRP8 (Arg180)/mock and MRP8 (Gly180)-mediated transportof cGMP, respectively.

328

9/30

1/50

G A
∆27 W

1/10

Figure 4 Worldwide frequency of allele A (open portion in each circle).
There is a north-south downward gradient from Northern China toward
Southern Asia with a peak in East Asians, and an east-west downward
gradient from Siberia toward Europe. Another mutation, a 27-bp deletion(D27) in exon 29 of ABCC11, independent of rs17822931 (538G-A), wasalso observed in subsets of individuals of Asian ancestry. Red numbers
indicate individuals with D27; #, US citizens of Northwest European
ancestry (from the CEPH families); *, US citizens of sub-Saharan Africanancestry (from the Coriell Institute for Medical Research).

auditory canal are its plausible functions2. The axillary odor is
known to be associated with wet-type cerumen and intensified bysweating. Histological study of the axillary skin from a patient withbromhidrosis showed numerous and large-sized apocrine glands22. In
nonhuman mammals, secretory products of apocrine sweat glands
such as body odor may have a role in sexual attraction as a sort ofpheromone. According to the genome sequence database and a PCR
search by us, the chimpanzee, orangutan, white-handed gibbon,
Japanese macaque and the dog have the orthologue of ABCC11, but
mice and rats lack it23. Instead, urinary odor types determined by the
major histocompatibility complex (MHC) have a similar biologicalrole in the rodents24. As ancient Northeast Eurasians are believed tohave lived in a cold environment, they might have acquired someselective advantages, such as less axillary odor or sweating, as an
adaptation to the cold climate.
METHODSSNP identification, genotyping, case-control study and haplotyping. Study
protocols were approved by the Institutional Review Boards of each institution
joining the study. Genomic DNA was extracted from whole blood of volunteers
using the standard method or from their fingernails with ISOHAIR (Nippon
Gene). Volunteers gave written informed consent. Other DNA samples werepurchased from the Coriell Institute for Medical Research. We searched for CA
repeat sequences and SNPs around the B5.9-cM segment containing D16S3093
and D16S311 and withdrew coding SNP (cSNP) data from the JSNP databasebecause the earwax type is a single-gene trait, and the JSNP data areconstructed from the Japanese, in which the dry type is known be the major

Arbitrary*a units 4

b

3

2

1

0
LLC-PK1-G

LLC-PK1-A

cGMP transport (pmol/mg protein)

G A
W W

mRNA expression(ABCC11/GAPDH)© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

rs17822931
Exon 29300
MRP8 (Gly180)

200
MRP8 (Arg180)
100
Mock

0

0

100

200
300
cGMP (µM)

400

500

VOLUME 38 [ NUMBER 3 [ MARCH 2006 NATURE GENETICS© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

LETTERSphenotype2. As we had previously found a shortage of SNPs in this region17,18,
we identified 15 new SNPs by sequencing the region among eight individuals
with wet earwax. A total of 134 CA repeat polymorphisms from the region and
15 new SNPs (three already deposited in dbSNP) as well as 21 registered SNPs
were used as markers for the first-step case-control studies by genotyping 118
Japanese volunteers (64 individuals with dry earwax and 54 controls with wet
earwax) whose DNA samples were collected in an anonymous manner andwhose earwax types were self-declared. We found two SNPs, B81540.1 and IMSJST141676, that were homozygous in 64 individuals with dry earwax. This led
us to focus on a B600-kb region between the two SNPs, especially on a fourgene (ABCC12, ABCC11, LONPL and SIAH1) interval, on which we performed
PCR-based genome sequencing of genomic DNA except on long repetitiveelements in the eight wet-type individuals. If a given SNP within a gene isclose to the earwax locus, these individuals are expected to show
80–90% heterozygosity, in view of the previously reported earwax phenotype
data in the Japanese2,4. In this interval, we found 37 SNPs (15 registered and
22 novel SNPs) and used them for another association study among the same
118 samples above and for the construction of LD blocks to confirm the 600-kb
region as a candidate region. LD blocks were constructed using the Haploview
program25. Because none of the SNPs above completely explained the earwax
phenotype as a mendelian trait, we collected blood from another series of 126
Japanese volunteers (88 dry-type and 38 wet-type individuals) from Nagasaki
City, whose earwax types were identified by a medical practitioner, and usedthem for the second-step association study.
Using the Haploview program25, we constructed haplotypes including
24 SNPs and D27 in the 600-kb interval and its flanking region in Native
Americans, Native Bolivians and Southern Han Chinese individuals. Primer
sequences for construction of haplotypes in various populations are listed in
Supplementary Table 4 online.
Complete genome sequencing of the individuals with dry earwax and wet
earwax. We adopted two strategies, PCR-based complete genome sequencing in eight wet-type individuals and BAC clone–based sequencing in a
wet/dry-type heterozygote for the genomic regions that could not be amplified
from the eight samples. Comparisons between the PCR-based sequenceand the complete contig sequence, and between two BAC clones containing
the allele responsible for the wet- or dry-type phenotype showed no structuralabnormality or any base changes other than SNPs found among the eight
wet-type individuals. The complete sequence consisted of 341,290 bp
and contained four genes (ABCC12, ABCC11, LONPL and SIAH1) spanningan LD block.
LLC-PK1 cell lines expressing allele A or G of human ABCC11, preparationof plasma membrane vesicles from these cells and detection of ATPdependent transport of cGMP. Full-length cDNA carrying allele A (dry-typeallele) and that carrying allele G (wet-type allele) of human ABCC11 wereinserted into pcDNA3.1-Hygro (Invitrogen) and transfected into pig-derived
LLC-PK1 cells (Health Science Research Resources Bank (HSRRB) cell bank)using Lipofectin reagent (Invitrogen). Total RNA was prepared from ABCC11expressing cell lines (LLC-PK1-A and LLC-PK1-G carrying alleles A and G,
respectively) and was used for reverse transcription (RT). Real-time PCR
quantification was carried out using a 7900HT Sequence Detection System(Applied Biosystems) as described previously26. Expression of ABCC11-mRNA
was compared with that of GAPDH-mRNA.
Plasma membrane vesicles were prepared from LLC-PK1 cells as described
previously27. The standard incubation medium contained the plasma membrane vesicles (50 mg protein), 500 mM 3H-labeled cGMP (Amersham),
250 mM sucrose, 10 mM Tris HCl (pH 7.4), 10 mM MgCl2, 1 mM ATP,
10 mM creatine phosphate and 100 mg ml–1 creatine kinase in a final volumeof 100 ml. Plasma membrane vesicles were incubated at 37 1C for 20 min with
3H-labeled cGMP at different concentrations (0, 5, 10, 20, 50, 100, 250 and
500 mM) in the absence (data not shown) or presence of 1 mM ATP. The
amount of 3H-labeled cGMP incorporated into the membrane vesicles wasmeasured by a rapid filtration technique28. ATP-dependent cGMP transport
was measured by the difference in the radioactivity incorporated into thevesicles in the presence and absence of ATP.

NATURE GENETICS VOLUME 38 [ NUMBER 3 [ MARCH 2006

URLs. Haploview program (http://www.broad.mit.edu/mpg/haploview/index.php); International HapMap Project (http://hapmap.jst.go.jp/index.
html.en); NCBI GenBank (http://www.ncbi.nih.gov/GenBank); JSNP database(http://snp.ims.u-tokyo.ac.jp); Coriell Institute for Medical Research (http://coriell.umdnj.edu/); dbSNP (http://www.ncbi.nih.gov/SNP/).
Accession codes. GenBank: 134 CA repeat polymorphisms, from AC003034 toAC007728. JSNP: 21 registered SNPs, from IMS-JST085980 to MS-JST141675.
dbSNP: newly identified SNPs used in this project, ss49784051 to ss49784077.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
We are grateful to all volunteers, especially to Nagasaki University Administration
Officers, for their participation in this study and are grateful to S. Horai,
P. Duanchang, S. Pookajorn, A. Soemantri, W. Settheetham-Ishida, P.G. Babu,
R. Kimura, P.N. Tongol-Rivera, K. Tajim, L.H. Sulaiman and K. Na-Bangchang
for assistance in collecting samples. We also thank N. Yanai and Y. Noguchi for
their technical assistance and S.M. Kennedy for correction of the manuscript. This
study was supported in part by CREST from the Japan Science and Technology
Agency (JST) and Grants-in-Aid for Scientific Research (Category S, No.
13854024; Priority Area ‘Applied Genomics’ No. 17019055; Category C, No.
17590288) from the Ministry of Education, Culture, Sports, Science andTechnology of Japan.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturegenetics
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
1. Adachi, B. Das Ohrenschmalz als Rassenmerkmal und der Rassengeruch (‘Achselgeruch’) nebst dem Rassenunterschied der Schweissdrüsen. Z Rassenk 6, 273–307(1937).
2. Matsunaga, E. The dimorphism in human normal cerumen. Ann. Hum. Genet. 25,
273–286 (1962).
3. Petrakis, N.L., Molohon, K.T. & Tepper, D.J. Cerumen in American Indians: Genetic
implications of sticky and dry types. Science 158, 1992–1993 (1967).
4. Omoto, K. Polymorphic traits in peoples of eastern Asia and the Pacific. Isr. J. Med.
Sci. 9, 1195–1215 (1973).
5. Petrakis, N.L., Pingke, U., Petrakis, S.J. & Petrakis, S.L. Evidence for a genetic cline
in earwax type in the Middle East and Southeast Asia. Am. J. Phys. Anthropol. 35,
141–144 (1971).
6. Petrakis, N.L. Cerumen genetics and human breast cancer. Science 173, 347–349(1971).
7. Ing, R., Petrakis, L. & Ho, H.C. Evidence against association between wet cerumen andbreast cancer. Lancet 1, 41 (1973).
8. Tomita, H.-a. et al. Mapping of the wet/dry earwax locus to the pericentromeric regionof chromosome 16. Lancet 359, 2000–2002 (2002).
9. Yabuuchi, H., Shimizu, H., Takayanagi, S. & Ishikawa, T. Multiple splicing variants of
two new human ATP-binding cassette transporters, ABCC11 and ABCC12. Biochem.
Biophys. Res. Commun. 288, 933–939 (2001).
10. Tammur, J. et al. Two new genes from the human ATP-binding cassette transportersuperfamily, ABCC11 and ABCC12, tandemly duplicated on chromosome 16q12.
Gene 273, 89–96 (2001).
11. Bera, T.K., Lee, S., Salvatore, G., Lee, B. & Pastan, I. MRP8, a new member of ABC
transporter superfamily, identified by EST database mining and gene prediction
program, is highly expressed in breast cancer. Mol. Med. 7, 509–516 (2001).
12. Guo, Y. et al. MRP8, ATP-binding cassette C11 (ABCC11), is a cyclic nucleotide efflux
pump and a resistance factor for fluoropyrimidines 2¢,3¢-dideoxycytidine and 9¢-(2¢phosphonylmethoxyethyl) adenine. J. Biol. Chem. 278, 29509–29514 (2003).
13. Chen, Z.S., Guo, Y., Belinsky, M.G., Kotova, E. & Kruh, G.D. Transport of bile acids,
sulfated steroids, estradiol 17-beta-D-glucuronide, and leukotriene C4 by humanmultidrug resistance protein 8 (ABCC11). Mol. Pharmacol. 67, 545–557 (2005).
14. Burkhart, C.N., Kruge, M.A., Burkhart, C.G. & Black, C. Cerumen composition by
flash pyrolysis-gas chromatography/mass spectrometry. Otol. Neurotol. 22, 715–722(2001).
15. Shugyo, Y. et al. Morphological differences between secretory cells of wet and dry typeof human ceruminous glands. Am. J. Anat. 181, 377–384 (1988).
16. Shulenin, S., Nogee, L.M., Annilo, T., Wert, S.E., Whitsett, J.A. & Dean, M. ABCA3
gene mutations in newborns with fatal surfactant deficiency. N. Engl. J. Med. 350,
1296–1303 (2005).
17. Diller, K.C., Gilbert, W.A. & Kocher, T.D. Selective sweeps in the human genome: A
starting point for identifying genetic differences between modern human and chimpanzees. Mol. Biol. Evol. 19, 2342–2345 (2002).
18. The International HapMap Consortium. A haplotype map of the human genome. Nature
437, 1299–1320 (2005).

329

LETTERS
24. Yamasaki, K., Singer, A. & Beauchamp, G.K. Origin, functions and chemistry of H-2
regulated odorants. Genetica 104, 235–240 (1999).
25. Barrett, J.C., Fry, B., Maller, J. & Daly, M.J. Haploview: analysis and visualization of LD
and haplotype maps. Bioinformatics 21, 263–265 (2005).
26. Masuzaki, H. et al. Detection of cell free placental DNA in maternal plasma: direct evidence
from three cases of confined placental mosaicism. J. Med. Genet. 41, 289–292 (2004).
27. Mitomo, H. et al. A functional study on polymorphism of the ATP-binding cassettetransporter ABCG12: Critical role of arginine-482 in methotrexate transport. Biochem.
J. 373, 767–774 (2003).
28. Ishikawa, T. et al. High-speed screening of human ABC transporter function andgenetic polymorphisms: New strategies in pharmacogenomics. Methods Enzymol.
400, 485–510 (2005).

© 2006 Nature Publishing Group http://www.nature.com/naturegenetics

19. Bonatto, S.L. & Salzano, F.M. A single and early migration for the peopling of theAmericas supported by mitochondrial DNA sequence data. Proc. Natl. Acad. Sci. USA
94, 1866–1871 (1997).
20. Tokunaga, K., Ohashi, J., Bannai, M. & Juji, T. Genetic link between Asians and native
Americans: Evidence from HLA genes and haplotypes. Hum. Immunol. 62,
1001–1008 (2001).
21. Dillehay, T.D. Tracking the first Americans. Nature 425, 23–24 (2003).
22. Bang, Y.H. et al. Histopathology of apocrine bromhidrosis. Plast. Reconstr. Surg. 98,
288–292 (1996).
23. Shimizu, H. et al. Characterization of the mouse Abcc12 gene and its transcript
encoding an ATP-binding cassette transporter, an orthologue of human ABCC12. Gene
310, 17–28 (2003).

330

VOLUME 38 [ NUMBER 3 [ MARCH 2006 NATURE GENETICS