cell biochemistry and function
Cell Biochem Funct 2009; 27: 402–405.
Published online 17 July 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/cbf.1582

SHORT COMMUNICATIONHERC1 polymorphisms: population-specific variations in
haplotype composition
Isao Yuasa 1*, Kazuo Umetsu 2, Hiroaki Nishimukai 3, Yasuo Fukumori 4, Shinji Harihara 5, Naruya Saitou 6,
Feng Jin 7, Prasanta K. Chattopadhyay 8, Lotte Henke 9 and Jürgen Henke 9
1

Division of Legal Medicine, Faculty of Medicine, Tottori University, Yonago, Japan
Department of Forensic Medicine, Faculty of Medicine, Yamagata University, Yamagata, Japan
3
Division of Legal Medicine, Ehime University School of Medicine, Ehime, Japan
4
Osaka Red Cross Blood Center, Osaka, Japan
5
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan
6
Division of Population Genetics, National Institute of Genetics, Mishima, Japan
7
Institute of Psychology, Chinese Academy of Sciences, Beijing, China
8
Amity Institute of Forensic Sciences, Defence Colony, New Delhi, India
9
Institut für Blutgruppenforschung, Cologne, Germany
2

Human HERC1 is one of six HERC proteins and may play an important role in intracellular membrane trafficking. The human HERC1 gene issuggested to have been affected by local positive selection. To assess the global frequency distributions of coding and non-coding single
nucleotide polymorphisms (SNPs) in the HERC1 gene, we developed a new simultaneous genotyping method for four SNPs, and applied thismethod to investigate 1213 individuals from 12 global populations. The results confirmed remarked differences in the allele and haplotype
frequencies between East Asian and non-East Asian populations. One of the three common haplotypes observed was found to be characteristicof East Asians, who showed a relatively uniform distribution of haplotypes. Information on haplotypes would be useful for testing the functionof polymorphisms in the HERC1 gene. This is the first study to investigate the distribution of HERC1 polymorphisms in various populations.
Copyright # 2009 John Wiley & Sons, Ltd.
key words — genotyping; haplotype; HERC1; single nucleotide polymorphism; population study

INTRODUCTION
The human HERC protein family consists of six proteins,which are characterized by the presence of a HECT (homologous
to E6-associated protein (UBE3A) carboxyl terminus)
domain and one or more RCC1 (regulator of chromosome
condensation)-like domains. The HECT domain is endowedwith the ability to act as ubiquitin ligases, and the RCC1-likedomain stimulates guanine nucleotide exchange on ADPribosylation factor 1 (ARF1) and on certain members of the
RAS oncogene (Rab) family of proteins, including Rab3A
and Rab5. HERC1 is an unusually large protein of 532 kDa
containing 4861 amino acids. HERC1 is widely expressed inmany tissues and is located in the cytosol and the Golgi
apparatus. HERC1 may play an important role in intracellular
membrane trafficking both in the cytoplasm and Golgi* Correspondence to: I. Yuasa, Division of Legal Medicine, Faculty of
Medicine, Tottori University, Yonago 683-8503, Japan. Tel: þ81-859-386123. Fax: þ81-859-38-6120. E-mail: yuasai@med.tottori-u.ac.jp

Copyright # 2009 John Wiley & Sons, Ltd.

apparatus.1 HERC1 also interacts with tuberous sclerosis 2(TSC2, tuberin), which suppresses cell growth, and results inthe destabilization of TSC2.2 The biological function of
HERC1, however, has not been well defined.
The human HERC1 gene (GenBank GeneID: 8925) spans
225 kb on chromosome 15q22 and consists of 78 exons. The
NCBI single nucleotide polymorphism (SNP) database lists
19 missense, 30 synonymous, and 7 frame shift mutations in
its exons. Very little is known about the associations between
the polymorphisms and functions of the HERC1 protein. A
few HERC1 polymorphisms in coding exons show remarked
differences in allele frequencies between East Asian
populations (Chinese and Japanese) and other populations(Africans and Caucasians). Recent studies using publicly
available datasets have suggested that these differences havebeen affected by positive selection.3–6 However, no
populations other than these four have been investigatedwith regard to the distribution of the polymorphisms in the
HERC1 gene. As an initial study toward a fuller understanding of HERC1 variation, we developed a novel
Received 15 April 2009
Revised 18 May 2009
Accepted 19 May 2009

403

HERC1 POLYMORPHISMS IN VARIOUS POPULATIONS

simultaneous genotyping method for four SNPs in the 30 half genomic region of the HERC1 gene, based on thequadruplex amplified product length polymorphism (APLP)
technique and investigated the distribution of genotypes in
12 global populations to obtain allele and haplotype
frequencies.
MATERIALS AND METHODS
Biological samples
DNA samples were obtained from 1213 unrelated individuals living in various areas of Eurasia and South America
and were used for a population study. Sub-Saharan African(Nigerian and Ghanaian) and Turk samples were collectedfrom people residing in Germany (Table 1). Most of thesesamples were from a set used in previous studies.7,8 DNA
samples from three chimpanzees were also investigated todetermine the ancestral haplotype. This study was approvedby the Ethical Committee at the Faculty of Medicine, Tottori
University.

showing relatively high heterozygosity values in the SNP
database, were selected as shown in Table 1. Three of themoccurred in the coding sequence, and one was located in an
intronic sequence. These SNPs were simultaneously
genotyped by quadruplex PCR based on the APLP
method.7–9 The nucleotide sequence and final concentrationof each primer are shown in Table 2. The PCR cocktail
consisted of 50 ml of Multiple PCR Master Mix from a
Multiplex PCR Kit (Qiagen, Hilden, Germany), 6.4 ml of 12
primers with a concentration of 100 pmol/ml, and 43.6 ml ofwater. 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
10–20 ng of genomic DNA. The cycle conditions were 958C
for 15 min; then 30 cycles of 948C for 10 s, 568C for 10 s,
728C for 10 s, and a final extension step of 15 min at 728C.
The products were separated using a polyacrylamide gel(9%T, 5%C) together with positive and negative controls
and then visualized by ethidium bromide staining.

Statistical analysis
Genotyping procedure
In the 19 missense mutations in the database, only three
SNPs are shown to have high heterozygosity values. Two
SNPs are rs2228510 and rs2228511 investigated in thisstudy. The other is rs36089909, but this was not polymorphic
in our preliminary study. Although several synonymous
mutations with frequency data are described in the 50
genomic region, they are less polymorphic. Finally, four
SNPs, rs2228510, rs2228511, rs2229749, and rs4523879

The estimation of allele and haplotype frequencies, testingof the Hardy–Weinberg equilibrium, and analysis ofmolecular variance (AMOVA) were carried out using theArlequin program ver. 3.11.10
RESULTS AND DISCUSSION
The APLP method requires three primers for the amplification of DNA fragments at a locus: two allele-specific primers
differing in length and one common primer for the opposite

Table 1. Four SNPs and allele frequencies in 12 global populations with data from the HapMap database

No.

Population

1
2
3
4
5
6
7
8
9
10
11
12

AfricanGermanTurk
IndianBuryatMongolian
Korean (Kwangju)
Japanese (Tottori)
Han (Wuxi)
Han (Huizhou)
Thai
ColumbianAfrican (YRI)
Caucasian (CEU)
Japanese (JPT)
Chinese (CHB)

Copyright # 2009 John Wiley & Sons, Ltd.

rs2228510

rs2228511

rs2229749

rs4523879

Exon 37

Exon 45

Exon 57

Intron 73

g.155692

g.172119

g.188939

g.210769

c.6806A>G

c.9241A>G

c.11166C>GINV73-289T>C

p.I2220V

p.P3031P

p.D3722En

A allele

G allele

G allele

C allele

68
92
80
107
98
102
120
104
119
111
114
98
59–60
58–60
43–45
45

0.53680.47830.65000.64950.90310.95590.95000.91830.93700.90540.92110.5459
0.608
0.342
0.911
0.956

0.0956
0.11410.27500.34110.84690.88240.92080.90870.89500.89640.87720.4745
0.169
0.043
0.907
0.933

0.0956
0.11410.27500.34110.84690.87750.91250.90870.89500.89190.86840.4745
0.183
0.050
0.900
0.911

0.0956
0.11410.27500.34110.84690.88240.92080.90870.89500.90090.88160.4745
0.178
0.043
0.898
0.933

Cell Biochem Funct 2009; 27: 402–405.
DOI: 10.1002/cbf

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i. yuasa ET AL.

Table 2. PCR primers for simultaneous typing of four SNPs in HERC1 gene and their final concentrations
SNP

Primer name

Sequence (50 ! 30 )

rs2228510

HERC1-E37F
HERC1-E37RA
HERC1-E37RG
HERC1-E45FA
HERC1-E45FG
HERC1-E45R
HERC1-E57F
HERC1-E57RG
HERC1-E57RC
HERC1-i73F
HERC1-i73RT
HERC1-i73RC

TCCAGGAGACCCTATTTGTAGTC
tataGTTCGGTGAAGGATtCGGATGTTCGGTGAAGGATAaGGAC
tatAATGTGGGAGTGGtAATCCA
ATGTGGGAGTGGAtATCCG
TCATGGCTAAGTACTTCTCCCTG
CAGACCAATGTGACTAGTGCAG
aatCCTGGCAATTTGATTCaTGC
CCTGGCAATTTGATTaCTGG
TTTCCAGCATTACCTAGTATCAGG
tattaGTAGTGATGTTCAAATCGtATCAAGTAGTGATGTTCAAATCGAtTCAG

rs2228511
rs2229749
rs4523879

Final concentration (mM)
0.2
0.2
0.2
0.4
0.4
0.4
0.2
0.2
0.2
1.2
1.6
1.2



Lowercase letters indicate non-complementary nucleotide.

DNA chain. Non-complementary nucleotides were introduced into the primers to produce a difference in length
between two PCR products, to enhance the specificity of theprimers, and to optimize the annealing temperature of theprimers.7–9 Figure 1 shows the band patterns of the products
obtained by quadruplex PCR. The nucleotide substitutions
were clearly and unambiguously detected as bands ofdifferent sizes. The sizes ranged from 55 bp for rs2229749C
to 100 bp for rs4523879T. Simultaneous genotyping by thepresent quadruplex PCR method is advantageous forinvestigating haplotypes because the amplicons at every
locus are obtained by only one amplification withoutsampling errors. In addition, this method is technicallysimple and inexpensive and does not require specialized
instruments.
We genotyped four SNPs in 1213 individuals from 12
global populations. The frequencies of an allele at each locusof the HERC1 gene are shown in Table 1. None of the
Hardy–Weinberg tests were significant. The allele frequencies in our African, German, Chinese, and Japanese samples
were similar to those in the SNP database. The SNPs at three

Figure 1. Simultaneous genotyping of the four SNPs by quadruplex PCR
based on the APLP method. Lanes M: 25-bp ladder; 1, 5, 6: haplotypes
AGGC; 2, 4, 9: GACT; 3, 10: GACT/AGGC; 7: AACT/AGGC; 8: AACT/GACT. The haplotypes estimated from the four SNPs are described in the
text and Table 3

Copyright # 2009 John Wiley & Sons, Ltd.

loci, rs2228511, rs2229749, and rs4523879, showedremarked differences in allele frequencies between theseven East Asian and five non-East Asian populations. The
frequencies for the derived alleles at these three SNP lociwere higher in the central part of East Asia than in thenorthern and southern parts.
Haplotypes were constructed on the basis of the genotype
data using the EM algorithm with phase-unknown samples,
and three main haplotypes (AACT, GACT, and AGGC in 50
to 30 order of the four SNPs) were observed in all populations
investigated here, indicating that the HERC1 protein wasclassified into three main types, Ile-Asp, Val-Asp, and IleGlu at the amino acid level. In addition, four minor
haplotypes (AGCC, GACC, GGCC, and GGGC) were also
observed at low or rare frequencies in some populations.
Thus, most of the polymorphisms were in strong linkage
disequilibrium, as evidenced by the low number of common
haplotypes. Three chimpanzees showed the same homozygous genotype, consisting of the AACT haplotype, which
was inferred to be an ancestral haplotype. Haplotypes GACTand AGGC arose by one and three mutation events,
respectively, from the ancestral haplotype AACT.
As shown in Table 3, the distribution of the three main
haplotypes was very diverse among various populations.
Haplotype AGGC prevailed mainly in the East Asian
populations, who showed relatively uniform frequencies, as
the frequencies ranged from 0.91 in Koreans to 0.85 in
Buryats. The Africans and Germans showed the lowest
frequencies whereas the Turks, Indians, and Columbians hadintermediate values. In contrast, the Africans and Germans
were characterized by fairly high frequencies of haplotypes
AACT and GACT. In the Columbians, the frequency of
haplotype AACT was as low as its frequencies in the East
Asian populations, but the frequency of haplotype GACT
was as high as its frequencies in Africans and Germans.
Haplotype heterozygosities were also calculated for theHERC1 locus. East Asian populations had a mean haplotype
heterozygosity of 0.2085. Haplotype heterozygosities weremuch higher outside of East Asia, with a mean value of
0.6162.
Cell Biochem Funct 2009; 27: 402–405.
DOI: 10.1002/cbf

405

HERC1 POLYMORPHISMS IN VARIOUS POPULATIONS

Table 3. Haplotype frequencies and heterozygosities in 12 global populations
No.

Population

n

AACT

GACT

AGGC

1
2
3
4
5
6
7
8
9
10
11
12

AfricanGermanTurk
IndianBuryatMongolian
Korean (Kwangju)
Japanese (Tottori)
Han (Wuxi)
Han (Huizhou)
Thai
Columbian

68
92
80
107
98
102
120
104
119
111
114
98

0.44120.36410.37500.32080.05610.07350.02920.00960.04200.01350.04390.0714

0.4632
0.52170.35000.33800.09690.04410.05000.08170.06300.08560.07460.4541

0.0956
0.11410.27500.32870.84690.87750.91250.90870.89500.89190.86840.4745

A high fixation index FST suggests positive selection.
AMOVA showed that the estimated FST value was as high as
0.401 among the 12 populations. Population pairwise FST
showed significantly differences between East Asian andnon-East Asian populations. No significant differences wereobserved between the Africans and Germans, between theTurks and Indians, or between the East Asian populations
except for between the Buryats and Koreans.
In this study, four SNPs of the HERC1 gene in 12 global
populations were simultaneously genotyped by the quadruplex PCR method. This is the first study to report on the
distribution of allele and haplotype frequencies in various
populations. This study confirmed that there are marked
differences in allele and haplotype frequencies between East
Asians and non-East Asians and showed that East Asians are
deficient in genetic diversity. The frequency of haplotype
AGGC has rapidly increased in East Asian populations.
Recently, some East Asian-specific alleles have been
demonstrated to be under local positive selection: ABCC11
is responsible for ear wax type11 and EDAR is associatedwith Asian hair thickness.12 The HERC1 protein is classified
into at least three main types of Ile-Asp, Val-Asp, and IleGlu. The frequencies of these types suggest the difference
between Asp and Glu at amino acid position 3722 to beimportant, but nothing is currently known regarding
the effect of the differences in amino acids on the functionof the HERC1 proteins. Information on haplotypes would be
useful in the design of biochemical experiments aimed at
testing the function of polymorphisms in the HERC1 gene.
These data will also be informative and facilitate genetic
association studies of HERC1-related diseases. Together
with the clarification of the significance of the mutations in
the HERC1 protein, the accumulation of data on theworldwide distribution of HERC1 polymorphisms using our
genotyping method may provide additional insights into therole of this protein and the remarked differences in the
distribution of its alleles and haplotypes.

Copyright # 2009 John Wiley & Sons, Ltd.

Others

0.01240.00490.00830.00900.0132

Heterozygosity
0.58590.58540.66540.67780.27150.22380.16460.16840.19410.19790.23930.5665ACKNOWLEDGEMENTS
This study was supported in part by a Grant-in-Aid for
Scientific Research (20590678 to IY) from the Japan Society
for the Promotion of Science.

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Cell Biochem Funct 2009; 27: 402–405.
DOI: 10.1002/cbf