Biochem Genet (2007) 45:535–542
DOI 10.1007/s10528-007-9095-9

Distribution of Two Asian-Related Coding SNPs in the
MC1R and OCA2 Genes
I. Yuasa Æ K. Umetsu Æ S. Harihara Æ A. Kido Æ A. Miyoshi Æ N. Saitou Æ
B. Dashnyam Æ F. Jin Æ G. Lucotte Æ P. K. Chattopadhyay Æ L. Henke Æ
J. Henke

Received: 23 January 2007 / Accepted: 2 March 2007 / Published online: 15 June 2007
Ó Springer Science+Business Media, LLC 2007

I. Yuasa (&)
Division of Legal Medicine, Faculty of Medicine, Tottori University, Yonago 683-8503, Japane-mail: yuasai@grape.med.tottori-u.ac.jp
K. Umetsu
Department of Experimental and Forensic Pathology, Faculty of Medicine, Yamagata University,
Yamagata, Japan
S. Harihara
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan
A. KidoDepartment of Legal Medicine, Faculty of Medicine, University of Yamanashi, Nakakoma, Japan
A. Miyoshi
Department of Forensic Medicine, Fukuoka University School of Medicine, Fukuoka, Japan
N. Saitou
Division of Population Genetics, National Institute of Genetics, Mishima, Japan
B. Dashnyam
Institute of Biological Sciences, Mongolian Academy of Sciences, Ulaan Baator, Mongolia
F. JinInstitute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
G. LucotteCenter of Molecular Neurogenetics, Paris, France
P. K. Chattopadhyay
Amity Institute of Forensic Sciences, Defence Colony, New Delhi, India
L. Henke  J. Henke
Institut für Blutgruppenforschung, Cologne, Germany

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Abstract Very little is known about the genes and mechanisms affecting skinlightening in Asian populations. In this study, two coding SNPs, c.G1129A (R163Q)
at the MC1R (melanocortin 1 receptor) gene and c.A1962G (H615R) at the OCA2
(oculocutaneous albinism type II) gene, were investigated in a total of 1,809 individuals in 16 populations from various areas. The Q163 and R615 alleles prevailed
almost exclusively in East and Southeast Asian populations. Wright’s FST was 0.445
for R163Q and 0.385 for H615R among the 16 populations. The frequency of the
Q163 allele was higher in Northeast Asians than in Southeast Asians. The frequencyof the R615 allele was highest in South China and unlikely to be associated withlevels of ultraviolet radiation. This allele may be a good marker to study the genetic
affinity among East Asians because of its restricted distribution and marked difference in allele frequency.
Keywords
Skin color

MC1R  OCA2  Pigmentation  Polymorphism  Population study Introduction
Skin color, one of the most visible signs of human biological variation, is markedly
diverse among populations. Skin color shows 88% of the total variation among
regions in contrast to classical genetic markers and DNA polymorphisms. The
global variation in skin color is strongly suggested to have been affected by natural
selection (Relethford 2002). Skin color among indigenous peoples is correlated withlevels of ultraviolet radiation and becomes lighter in more northerly latitudes(Jablonski and Chaplin 2000). In spite of many candidate genes, relatively little isunderstood about the genetic and molecular basis of human skin variation. Recently,
L374F in the membrane-associated transporter protein (SLC45A2 gene) and A111T
in a putative cation exchanger protein (SLC24A5 gene) were found to have a unique
distribution in allele frequency: the mutant alleles prevail extensively in Europeans,
whereas the wild-type alleles are fixed in Africans and Asians. These two mutant
alleles show strong evidence of having undergone positive selection in Europeans(Graf et al. 2005; Lamason et al. 2005; Nakayama et al. 2002; Soejima et al. 2006;
Yuasa et al. 2004, 2006). Asian populations also have light skin, for which no genes
are known to be responsible, although several candidate genes have been suggested(McEvoy et al. 2006; Myles et al. 2007).
Melanocortin 1 receptor (MC1R), belonging to a family of G protein-coupled
receptors, leads to the production of eumelamin after the binding of a-melanocytestimulating hormone (a-MSH). Some mutations in the MC1R gene on chromosome
16q24.3 are associated with red hair, fair skin, and freckles (Valverde et al. 1995).
The R163Q (CGA?CAA, refSNP ID: rs885479) variant is observed at highfrequencies in East and Southeast Asians and Native Americans (Makova and
Norton 2005; Nakayama et al. 2006; Rana et al. 1999). Although the Q163 allele
had slightly less affinity for a-MSH in binding and second-messenger studies, thismutation was not considered to have an important role in supporting the red-haired
phenotype (Ringholm et al. 2004). The OCA2 gene (P-related gene) on chromosome

123Biochem Genet (2007) 45:535–542

537

15q11.2–q12, the human homolog of the murine pink-eyed dilution (p) gene, plays arole in regulating the pH of melanosomes. Some mutations in the OCA2 gene resultin oculocutaneous albinism type II (Oetting et al. 2005; Puri et al. 2000). The
H615R (CAT?CGT, refSNP ID: rs1800414) mutation at the OCA2 locus issuggested to be Asian-specific (Lee et al. 1995). However, there have been few
studies of the H615R mutation. We therefore investigated the frequency of theH615R polymorphism in several populations as well as that of the R163Q
polymorphism in the MC1R gene.

Materials and Methods
DNA samples
DNA samples were obtained from 1809 unrelated individuals, mainly living invarious areas of Eurasia. The donors were selected at random, irrespective of skincolor, and their phenotypic data were unavailable. All samples except two African
population samples were the same set as used in our previous study (Yuasa et al.
2006). The African samples were collected in Germany.
Simultaneous Typing of the Two Coding SNPs
DNA samples were typed simultaneously for the two polymorphisms on the basis of
the amplified product length polymorphism (APLP) method (Watanabe et al. 1997;
Yuasa et al. 2004). A PCR cocktail consisted of 100 ml of Multiple PCR Master Mix
in Multiplex PCR Kit (Qiagen), 92 ml of six primers with a concentration of
10 pmol/ml, and 8 ml of water. The nucleotide sequence and amount of each primer
are shown in Table 1. PCR was performed in a volume of 8 ml containing 7.5 ml of
the PCR cocktail and 0.5 ml of a solution containing about 20 ng of genomic DNA.
Cycle conditions were 958C for 15 min, then 30 cycles of 948C for 30 s, 568C for
30 s, 728C for 40 s, and a final extension step of 10 min at 728C, in a GeneAmp PCR
System 9700 (Applied Biosystems, Foster City, CA). The products were separated

Table 1 PCR primers for simultaneous typing of the MC1R R163Q and OCA2 H615R polymorphisms
and their amounts in 200 ml of PCR cocktail
Name

Sequence (50 ?30 ) a

Amount (ml)MC1R-163F

aCTCCATCTTCTACGCACTGCG

16

MC1R-163RA

tttaGATGGCCGCAACaGCTT

20

MC1R-163RG

GATGGCCGCAACGaCTC

16

OCA2-615FG

attaCTGTGGTTTCTCTCTTACAtCG

16

OCA2-615FA

CTGTGGTTTCTCTCTTACtGCA

12

OCA2-615R

CATTTGGCGAGCAGAATCC

12aLowercase letters indicate noncomplementary nucleotides

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Biochem Genet (2007) 45:535–542

using a polyacrylamide gel (9%T, 5%C), then visualized by ethidium bromide
staining.
Statistical Analysis
Allele frequencies were estimated and Hardy–Weinberg equilibrium was tested
using Arlequin program ver. 3.01 (Excoffier et al. 2005). Wright’s FST statistic wasused to estimate the proportion of variation attributable to differences in the R163Q
and H615R mutations among 16 populations (Hartl and Clark 1997).

Results and Discussion
For the amplification of DNA fragments at a locus, the APLP method requires three
primers: two allele-specific primers differing in length and one common primer on
the opposite DNA chain. Figure 1 shows the banding patterns of PCR products
obtained by simultaneous amplification of the two coding SNPs. Each genotype wasclearly and unambiguously distinguished. The amplicons were generated only when
the two allele-specific primers matched 30 ends and the nucleotide substitutions weredetected as amplified fragments with different lengths.
Table 2 summarizes differences in the distribution of the genotypes and allele
frequencies for the MC1R and OCA2 genes in a total of 1809 unrelated individuals
from 16 populations in three regions. In each population, there was no statisticallysignificant departure from Hardy–Weinberg equilibrium (data not shown).
Our data on the distribution of the Q163 allele at the MC1R locus were notinconsistent with those reported previously (Makova and Norton 2005; Nakayama
et al. 2006; Rana et al. 1999). Wright’s FST was estimated to be 0.445 among the 16
populations investigated in this study. The Q163 allele was observed at highfrequencies in all eight Asian populations. The Japanese population in Okinawa had
the highest frequency of 0.833 which was comparable to 0.85 in Ainu (Nakayama
et al. 2006) and Tibet (Shi et al. 2001). The Q163 allele is very common in New
World populations, which have an affinity with East Siberian and Northeast Asian
populations (Harding et al. 2000; Rana et al. 1999). The frequency of this allele isFig. 1 Simultaneous genotyping of the R163Q and H615R polymorphisms. Lane L: 25-bp ladder. Lanes
1–6: Japanese. Lanes 7–12: Germans

123Han Chinese

Han Chinese

Han ChineseIndonesian

13

14

15

16

Surabaya

Huizhou

Wuxi

Shenyang

Okinawa

143

105

111

119

87

87

103

a

HapMap data, not included in calculation of Wright’s FST

Wright’s FST

60

Japanese

12

Tottori

Eastern Mongolia

YRIa

Japanese

11

173

20

Buryat10

Ulaan Baator

118

107

44

Khalha9

Dhaka

New Delhi

JPTa

Bangladeshi

8

98
200

19

Indian7West Germany

Rheims

45

Turk

6

92

199

60

French5

MunichNorthrhine-Westphalia

CEU

German

4

31
36

CHBa

German3

Northrhine-Westphalia

Northrhine-Westphalia

17

Ghanaian2n18

Nigerian1

Collection placea

Population

No.

Table 2 Distribution of two SNPs in the MC1R and OCA2 genes

31

23

17

19

7

3

4

13

13

88

96

172

92

79

188

36

0

no datano datano datano data

54

62

50

39

23

45

62

72

29

10

26

6

12

10

0

0

28

32

50

41

61

54

68

88

1

1

2

0

1

1

0

31

60

8

7

60

84

18

19

26

24

17

107

109

116

107

194

98

92

198

36

0

26

19

0

20

46

64

44

44

59

34

57

2

0

6

0

0

1

0

0

H/R

H

R

Q

Genotypes of H615R

Genotypes of R163Q
R/Q

OCA2:

MC1R:

0

10

19

0

1

47

36

17

19

27

2

7

0

0

0

0

0

0

0

0

R

0.445

–

–

–

–

0.524

0.568

0.630

0.695

0.833

0.743

0.692

0.717

0.131

0.056

0.075

0.031

0.076

0.030

0.000

0.000

MC1R Q163

Allele frequency

0.385

0.000

0.523

0.633

0.000

0.105

0.630

0.571

0.448

0.471

0.549

0.133

0.205

0.009

0.000

0.015

0.000

0.000

0.003

0.000

0.000

OCA2 R615

Biochem Genet (2007) 45:535–542
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Biochem Genet (2007) 45:535–542

rather low in Han Chinese in Huizhou and Indonesians in Surabaya. A previous
study of 30 Asian and Oceanian populations revealed that East Asians apparently
had high frequencies (0.70 on average) in comparison with Southeast Asians (0.44)
and Oceanians (0.01) (Nakayama et al. 2006).
The R615 allele at the OCA2 locus was rare or not found in Indo-European and
African populations, whereas it was very common in Asian populations. Wright’s
FST for the R615 allele was estimated to be 0.385 among the 16 populations and was
lower than that for the Q163 allele at the MC1R locus. The R615 allele had adifferent distribution from the Q163 allele and was most common in Huizhou(0.63). Japanese had similar or somewhat low frequencies in comparison with Han
Chinese in Wuxi and Huizhou. Khalhas, Buryats, and Indonesians in thesurrounding areas showed the lowest frequencies among East Asian populations.
Frequencies of the R615 allele in the populations from the International HapMap
Consortium (http://www.hapmap.org/) are consistent with findings of the present
study (see Table 2). The R615 allele is likely to have occurred more recently thanthe Q163 allele which was estimated to have arisen after the divergence of Asians,
Europeans, and Africans and before the divergence of Asians and Native Americans(Rana et al. 1999) and 100,000–250,000 years ago (Harding et al. 2000). The R615
allele must have spread rapidly to Asian populations after its occurrence in China.
Its distribution is not associated with ultraviolet radiation levels, and it is unlikely tocontribute to the light skin of Mongolians and Buryats. The R615 allele may be agood marker to study the genetic affinity among East Asians because it is nearly
restricted to them and its frequency is extremely different among them.
No genes are known to be responsible for light skin in Asian populations.
Recently, McEvoy et al. (2006) proposed several candidate genes, functionally
important for skin pigmentation, in their model for the evolutionary geneticarchitecture of human pigmentation in the three major human populations. The
MC1R and OCA2 genes, showing relatively strong signatures for natural selection,
are also included among the candidate genes affecting skin lightening in East Asian
populations. Similarly, Myles et al. (2007) used both a long-range haplotype test
and an FST-based approach of pigmentation genes and identified the DCT(dopachrome tautomerase, or tyrosine-related protein 2) gene as a candidate genefor recent positive selection in the Chinese. However, no nonsynonymous SNPs
have been found within the DCT gene to date. Interaction between the MC1R and
OCA2 genes may be important in skin pigmentation. Akey et al. (2001) genotyped
three SNPs in the MC1R gene and two SNPs in the OCA2 gene in a Tibetan
population and identified a significant interactive effect of V92M in the MC1R geneand IVS13–15 in the OCA2 gene on skin pigmentation. These findings suggest thatpolymorphisms in introns may also be important for skin pigmentation. According
to the HapMap datasets, there are some nearly Asian-specific mutations (such as
rs12438490, rs12440330, rs12442916, rs1545397, rs12439410, rs12440423, etc.) in
intronic regions of the OCA2 gene. These mutations are observed at extremely highfrequencies only in Chinese and Japanese. Further comprehensive analysis
including the study of polymorphisms in introns will be needed to understandskin pigmentation in Asians.

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References
Akey JM, Wang H, Xiong M, Wu H, Liu W, Shriver M, Jin L (2001) Interaction between themelanocortin-1 receptor and P genes contributes to inter-individual variation in skin pigmentation
phenotypes in a Tibetan population. Hum Genet 108:516–520
Excoffier L, Laval G, Schneider S (2005) Arlequin ver 3.0: an integrated software package for population
genetics data analysis. Evol Bioinform Online 1:47–50
Graf J, Hodgson R, van Daal A (2005) Single nucleotide polymorphisms in the MATP gene areassociated with normal human pigmentation variation. Hum Mutat 25:278–284
Harding RM, Healy E, Ray AJ, Ellis NS, Flanagan N, Todd C, Dixon C, Sajantila A, Jackson IJ, BirchMachin MA, Rees JL (2000) Evidence for variable selective pressures at MC1R. Am J Hum Genet
66:1351–1361
Hartl DL, Clark AG (1997) Principles of population genetics 3rd edn. Sunderland, Sinauer Associates
Jablonski NG, Chaplin G (2000) The evolution of skin coloration. J Hum Evol 39:57–106
Lamason RL, Mohideen MA, Mest JR, Wong AC, Norton HL, Aros MC, Jurynec MJ, Mao X,
Humphreville VR, Humbert JE, Sinha S, Moore JL, Jagadeeswaran P, Zhao W, Ning G,
Makalowska I, McKeigue PM, O’Donnell, Kittles R, Parra EJ, Mangini NJ, Grunwald DJ, Shriver
MD, Canfield VA, Cheng KC (2005) SLC24A5, a putative cation exchanger, affects pigmentation inzebrafish and humans. Science 310:1782–1786
Lee S-T, Nicholls RD, Jong MTC Fukai K, Spritz AS (1995) Organization and sequence of the human P
gene and identification of a new family of Transport proteins. Genomics 26:354–363
Makova K, Norton H (2005) Worldwide polymorphism at the MC1R locus and normal pigmentation
variation in humans. Peptides 26:1901–1908
McEvoy B, Beleza S, Shriver MD (2006) The genetic architecture of normal variation in humanpigmentation: an evolutionary perspective and model. Hum Mol Genet 15:R176–R181
Myles S, Somel M, Tang K, Kelso J, Stoneking M (2007) Identifying genes underlying skin pigmentationdifferences among human populaions. Hum Genet 120:613–621
Nakayama K, Fukamachi S, Kimura H, Koda Y, Soemantri A, Ishida T (2002) Distinctive distribution of
AIM1 polymorphism among major human populations with different skin color. J Hum Genet
47:92–94
Nakayama K, Soemantri A, Jin F Dashnyam B, Ohtsuka R, Duanchang P, Isa MN, Settheetham-Ishida W,
Harihara S, Ishida T (2006) Identification of novel functional variants of the melanocortin 1 receptor
gene originated from Asians. Hum Genet 119:322–330
Oetting WS, Garrett SS, Brott M, King RA (2005) P gene mutations associated with occulocutaneousalbinism type II (OCA2) Hum Mutat 25:323
Puri N, Gardner JM, Brilliant MH (2000) Aberrant pH of melanosomes in pink-eyed dilution (p) mutant
melanocytes. J Invest Dermatol 115:607–613
Rana BK, Hewett-Emmett D, Jin L Chang BH, Sambuughin N, Lin M, Watkins S, Bamshad M, Jorde LB,
Ramsay M, Jenkins T, Li WH (1999) High polymorphism at the human melanocortin 1 receptor
locus. Genetics 151:1547–1557
Relethford JH (2002) Apportionment of global human genetic diversity based on craniometrics and skincolor. Am J Phys Anthropol 118:393–398
Ringholm A, Klovins J, Rudzish R, Phillips S, Rees JL, Schioth HB (2004) Pharmacologicalcharacterization of loss of function mutations of the human melanocortin 1 receptor that areassociated with red hair. J Invest Dermatol 123:917–923
Shi P, Lu XM, Luo HR, Xiang-Yu J, Zhang YP (2001) Melanocortin-1 receptor gene variants in four
Chinese ethnics populations. Cell Res 11:81–84
Soejima M, Tachida H, Ishida T, Sano A, Koda Y (2006) Evidence for recent positive selection at thehuman AIM1 locus in a European population. Mol Biol Evol 23:179–188
Valverde P, Healy E, Jackson I, Rees JL, Thody AJ (1995) Variants of the melanocyte-stimulating
hormone receptor gene are associated with red hair and fair skin in humans. Nat Genet 11:328–330
Watanabe G, Umetsu K, Yuasa I, Suzuki T (1997) Amplified product length polymorphism (APLP): a
novel strategy for genotyping the ABO blood group. Hum Genet 99:34–37
Yuasa I, Umetsu K, Watanabe G, Nakamura H, Endoh M, Irizawa Y (2004) MATP polymorphisms inGermans and Japanese: L374F mutation as a population marker for Caucasoids. Int J Legal Med
118:364–366

123

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Biochem Genet (2007) 45:535–542

Yuasa I, Umetsu K, Harihara S, Kido A, Miyoshi A, Saitou N, Dashnyam B, Jin F, Lucotte G,
Chattopadhyay PK, Henke L, Henke J (2006) Distribution of the F374 allele of the SLC45A2(MATP) gene and founder-haplotype analysis. Ann Hum Genet 70:802–811

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