European Journal of Human Genetics (2009) 17, 793 – 801& 2009 Macmillan Publishers Limited All rights reserved 1018-4813/09 $32.00
www.nature.com/ejhg

ARTICLE

Variable number of tandem repeat polymorphismsof DRD4: re-evaluation of selection hypothesis andanalysis of association with schizophrenia
Eiji Hattori1,4, Mizuho Nakajima1,4, Kazuo Yamada1, Yoshimi Iwayama1, Tomoko Toyota1,
Naruya Saitou2 and Takeo Yoshikawa*,1,3
1

Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Saitama, Japan; 2Division of Population Genetics,
National Institute of Genetics, Shizuoka, Japan; 3CREST, Japan Science and Technology Agency, Tokyo, Japan

Associations have been reported between the variable number of tandem repeat (VNTR) polymorphisms
in the exon 3 of dopamine D4 receptor gene gene and multiple psychiatric illnesses/traits. We examinedthe distribution of VNTR alleles of different length in a Japanese cohort and found that, as reported earlier,the size of allele ‘7R’ was much rarer (0.5%) in Japanese than in Caucasian populations (B20%). This
presents a challenge to an earlier proposed hypothesis that positive selection favoring the allele 7R has
contributed to its high frequency. To further address the issue of selection, we carried out sequencing of
the VNTR region not only from human but also from chimpanzee samples, and made inference on theancestral repeat motif and haplotype by use of a phylogenetic analysis program. The most common 4R
variant was considered to be the ancestral haplotype as earlier proposed. However, in a gene tree of VNTR
constructed on the basis of this inferred ancestral haplotype, the allele 7R had five descendent haplotypes
in relatively long lineage, where genetic drift can have major influence. We also tested this length
polymorphism for association with schizophrenia, studying two Japanese sample sets (one with 570 casesand 570 controls, and the other with 124 pedigrees). No evidence of association between the allele 7R andschizophrenia was found in any of the two data sets. Collectively, this study suggests that the VNTR
variation does not have an effect large enough to cause either selection or a detectable association withschizophrenia in a study of samples of moderate size.
European Journal of Human Genetics (2009) 17, 793–801; doi:10.1038/ejhg.2008.247; published online 17 December 2008

Keywords: DRD4; VNTR; selection; phylogenetic network; schizophrenia

Introduction
Disturbances in the dopamine neurotransmitter systemhave long been suggested to play a crucial role in thepathogenesis of schizophrenia.1 However, involvement of
dopamine-related genes in the development of this*Correspondence: Dr T Yoshikawa, Laboratory for Molecular Psychiatry,
RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-city, Saitama 351-0198,
Japan. Tel: þ 81 48 467 5968; Fax: þ 81 48 467 7462;
E-mail: takeo@brain.riken.jp
4
The first two authors equally contributed to this work.
Received 5 August 2008; revised 6 October 2008; accepted 20 November
2008; published online 17 December 2008

disorder has remained elusive. The dopamine D4 receptor
gene (DRD4), located on chromosome 11p15.5,2,3 hasreceived considerable interest because clozapine, a neuroleptic, which is often effective for treatment-resistant
symptoms, has a high affinity to this receptor.4,5 Also, D4
receptor is upregulated in the postmortem brain tissues
from schizophrenic patients.6 – 9 To investigate genetic
association of DRD4 with psychiatric phenotypes andtraits, many studies have focused on a 48-bp variable
number of tandem repeat (VNTR) in exon 3. This tandem
repeat varies in length, comprising two (2R) to eleven (11R)
48-bp repeat units, and codes for the third intracellularDRD4: selection and association with schizophrenia
E Hattori et al794
loop of the receptor protein.10 The 7R variant protein has
functional properties distinct from other size variants interms of clozapine binding10 and effect on post-synaptic
intracellular signal transduction.11,12 Also, there has been
growing literature reporting genetic association of theallele 7R with psychiatric traits/illnesses including novelty
seeking,13,14 attention-deficit hyperactivity disorder(ADHD)15,16 as well as schizophrenia.13,17 – 31 Some studies
declared association of the VNTR size polymorphisms
with schizophrenia,13,17,21,22,24,27 although not all studies
agree.18,23,25,26,28,30 – 33
Also, supporting the functional role of the VNTR isevidence of selection favoring the allele 7R of the VNTR.34,35
It was shown that the ratio of non-synonymous (Ka) tosynonymous (Ks) substitutions, Ka/Ks, is higher than 1 in thistandem repeat regions, and that strong linkage disequilibrium (LD) exists between the allele 7R and the surrounding
DRD4 polymorphisms. Further, on the basis of inferred genegenealogy of VNTR alleles, the allele 7R was supposed to haveoriginated as a rare event involving multiple mutations andgene conversions. The observed high frequency of the allele
7R in many European ancestry populations despite acomplicated mechanism of its origin added support to thenotion of selection and functional importance of the VNTR.
In the world-wide populations, 35 sequence variants of the
48-bp repeat unit are known to exist, each of which isreferred to as a ‘motif’. To date, 56 different VNTR
haplotypes, with each being composed of an unique
combination of motifs, have been reported in humans.35
However, no study has ever exploited the data on sequencevariation both from human and chimpanzee samples forprocedures of population genetics analysis.
In this study, we attempted to make precise inference onancestral haplotype to construct a gene tree of VNTR by
subcloning and sequencing both human and chimpanzee
samples. We constructed a phylogenetic network of motifs
using an in-house computer program and inferred apossible genetic relationship of the VNTR haplotypes.
These analyses allowed us to re-evaluate the proposed
notion of selection acting on this gene locus. We alsostudied the VNTR for association with schizophrenia inJapanese case-control and pedigree sample sets.
In this study, the term ‘allele’ means the length variantof VNTR. The same length does not necessarily reflect thesame VNTR sequence as is shown in studies including thisone (see below in the Results section). A ‘motif’ refers to asequence variant of the 48-bp repeat unit of the VNTR.
Also, we mean a VNTR sequence or a motif composition by
the term ‘haplotype’.

Materials and methods
Length-analysis, subcoloning and re-sequencing of the
VNTR region
The VNTR locus was amplified by polymerase chain
reaction (PCR) using fluorescent-labeled primers. PCR was
European Journal of Human Genetics

carried out using an ABI 9700 thermocycler (Applied
Biosystems, Foster City, CA). PCR fragments were analyzed
on an ABI PRISM 3700 Genetic Analyzer (Applied Biosystems) and the genotype, or the constitution of size alleles,of each individual sample was determined using GeneScan
3.5.2 and Genotyper 3.6 software (Applied Biosystems).
Subsequently, the VNTR sequences of 102 human chromosomes from control subjects as well as 20 chimpanzee(Pan troglodytes) chromosomes were determined. We
carried out subcloning of a PCR product encompassing
the entire VNTR region using TOPO TA Cloning kit(Invitrogen, Carlbad, CA) and subsequently direct sequencing using the DYEnamic ET terminator cycle sequencing
kit (Amersham Biosciences, Piscataway, NJ) and the
ABI PRISM 3730 Genetic Analyzer (Applied Biosystems).
Sequences were aligned by the SEQUENCHER program(Gene Codes Corporation, Ann Arbor, MI).
The primer sequences and detailed information on thereaction conditions are available upon request.

Phylogenetic network analysis
Phylogenetic networks of human and chimpanzee motifs
were constructed as earlier described36,37 using an in-housecomputer program. Possible unequal crossing-over andnucleotide substitution events in the evolutionary history
were inferred.
Subjects for a case-control study
For case-control association analysis, samples from 570
unrelated cases of schizophrenia (285 men, 285 women;mean age 47.0±11.4 years), and 570 age- and sex-matched
controls (285 men, 285 women; mean age 46.7±11.1
years) were analyzed.38 Furthermore, for family-based
association test, we studied 124 pedigree sample sets with
376 members, of whom 163 were affected. This included 80
independent and complete trios (schizophrenic offspring
and their parents), 15 probands with one parent, 13
probands with affected siblings, and 30 probands withdiscordant siblings.39 Probands consisted of 72 males and
52 females.
The diagnosis of schizophrenia was made by consultation according to DSM-IV criteria with consensus from atleast two experienced psychiatrists. All available medical
records were taken into consideration. Control subjects
were recruited from hospital staffs and volunteers who
showed no evidence of psychoses during brief interviews
with psychiatrists. All subjects were from central Japan.
The study was approved by the Ethics Committee of
RIKEN, and all participants provided written informed
consent.
Statistical tests for association
In case-control analysis, the allelic and genotypic distributions were tested for association by a Monte-Carlotest as implemented in CLUMP program40 (number ofDRD4: selection and association with schizophrenia
E Hattori et al795
simulations and the random number seed set to 10 000 and
100, respectively). Fisher’s exact test was also performed totest each individual allele for association. In the analysis
of pedigree samples, transmission disequilibrium test was
performed to test for global association and for individual
allele association using PDT41 and FBAT (http://www.biostat.
harvard.edu/~fbat/default.html) software, respectively.

Supplementary Information), giving rise to three different
VNTR haplotypes (Table 3). One haplotype was novel but
all of the seven constituent motifs were included in theearlier report.43
To obtain more information on the sequence diversity
around the VNTR, we subsequently studied a fraction ofhuman and chimpanzee samples, and examined theadjacent two SNPs, rs1870723 (G/A) and rs7482904 (G/C)
located at 165-bp and 185-bp downstream of the VNTR,
respectively (Table 4). A haplotype, G – G, was in synteny
with most VNTR haplotypes including the most common
4R (H1 – H2 – H3 – H4) haplotype, whereas G – C haplotype
were found syntenic with all the 6R and 7R haplotypes.
Another haplotype, A – C, was observed in majority of 2R
haplotypes and one 4R (H1 – H2 – H13 – H4) haplotype. All
the chromosomes from chimpanzee samples had the G – G
haplotypes.

Results
Re-sequencing of the entire VNTR region
Seven length variants of the VNTR (2R, 3R, 4R, 4.5R, 5R,
6R, 7R) were detected (Table 1). We then carried out
subcloning for 102 human chromosomes so that all thesize alleles can be sequenced. Eleven motifs and 14 VNTR
haplotypes were identified (Table 2 and Supplementary
Table S1 in Supplementary Information). We named thehuman motifs H1 to H35. Three haplotypes were newly
identified in this study (Table 2). The frequency of theallele 7R in the Japanese cohort was extremely low (0.5%),in contrast to that of world-wide average (19.2%)(Table 1).35 The allele 4.5R was recently named so becauseof its size between 4 and 5 repeats.42 We identified thislength variant in eight chromosomes (four chromosomesin each of cases and controls). We re-sequenced six of thesechromosomes, and it revealed an insertion of an 18-bp
sequence, completely identical with the 30 immediate
downstream sequence of the VNTR into the middle portionof the most common 4R haplotype (Figure 1). We excluded
the possibility that the 4.5R may be an artifact that was
generated from the PCR and/or subcloning processes,
based on the results that allele compositions (eg genotypes)
in all the samples containing 4.5R allele were perfectly
consistent between the results from GeneScan analysis andthose from subcloning analysis. In addition, we did notdetect any novel amplicons except for the 4.5R in all theclones that we picked up and sequenced.
To add information for inference of an ancestral motif/
haplotype, 20 chimpanzee chromosomes were also
analyzed by subcloning. Chimpanzee VNTRs from all thechromosomes were the same in size, each consisting of fiverepeats of a 48-bp unit. There were seven sequence variants(motifs) of a 48-bp unit (Supplementary Table S1 in

Table 1Phylogenetic analysis for the VNTR
Human and chimpanzee VNTR haplotypes are composedof various motifs (Supplementary Table S1 in Supplementary Information) and their phylogenetic relationshipshould be quite complex. The earlier43 and current studies
detected a total of nine chimpanzee motifs, which we
designated as C1 to C9 (for the sequences of C8 and C9, see
Supplementary Table S2 in Supplementary Information).
Among the human motifs, H1, H2, H3, and H4 were mostfrequent and thus are likely to have constituted anancestral haplotype. To further corroborate this inference,
we conducted a phylogenetic network analysis to reveal
relationships among these four human motifs and all thenine chimpanzee motifs. Figure 2 shows the phylogenetic
network for those 13 motifs. C1 and C2 differ by only one
nucleotide, and they are closest to H1. C3, C4, C5, C8, andC9 are similar with each other, and they are closest to H2.
Remaining two chimpanzee motifs (C6 and C7) differ by
only one nucleotide, and they are closest to H4. There is no
chimpanzee motif that is close to H3. Three black circles in
Figure 2 indicate locations of common ancestral sequences
for human and chimpanzee, suggesting orthologous
relationships. It seems that the common ancestor ofhuman and chimpanzee possessed a haplotype similar toH1 – H2 – H3 – H4. If this scenario is true, chimpanzee

Distribution of the VNTR lengths in case (schizophrenia)-control samples

Allele

2R

3R

4R

4.5R

5R

6R

7R

Schizophrenia (N ¼ 1120)

0.119

0.003

0.830

0.004

0.036

0.007

0.002

Control (Na ¼ 1136)

0.114

0.012

0.821

0.004

0.036

0.007

0.005

a

Global P*

7R vs others**

0.166

0.163

VNTR, variable number of tandem repeat.
*P-value by Monte-Carlo test.
**P-value by Fisher’s exact test.
a
N represents the number of chromosomes.

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796

Table 2

Motif compositions of the 48-bp VNTR haplotypes (human)

AlleleFrequency

2R
3R

0.114
0.012

26
8

0.821

38

0.004
0.036
0.007

3
13
8

0.005

6

Total

102

4R
4.5R
5R
6R

b

7R

Motif compositiona

N
26
6
1
1
36
2
3
13
5
2
1
3
2
1

Haplotype frequency

H1 – H4
H1 – H2 – H4
H1 – H2 – H31
H1 – H11 – H4c
H1 – H2 – H3 – H4
H1 – H2 – H13 – H4
H1 – H2 – (18-bp) – H3 – H4
H1 – H11 – H2 – H3 – H4
H1 – H2 – H6-H5 – H2 – H20
H1 – H2 – H6 – H5 – H2 – H4
H1 – H2 – H3 – H2 – H3 – H4
H1 – H2 – H6 – H5 – H2 – H5 – H4
H1 – H2 – H13 – H5 – H2 – H5FH4c
H1 – H2 – H27 – H5 – H2 – H5 – H4c

0.114
0.009
0.002
0.002
0.778
0.043
0.004
0.036
0.004
0.002
0.001
0.003
0.002
0.001

VNTR, variable number of tandem repeat.
aNumbers represent motifs.
b
The 4.5R refers to an allele with a length between 4 and 5 repeats. See Figure 1 for details.
cNovel haplotypes identified in the present study.

18 bp

4R
1

2

3

4

5'

CCCCGACGCCGTCAGAGCCGCCGCGCKCCCAACCCCAGACTCCACCGCAA

3'

4.5R
1

CCCCGACGCCGTCAGAGC

2

5'

3

4

CCCCGACGCCGTCAGAGCCGCCGCGCKCCCAACCCCAGACTCCACCGCAA

3'

Figure 1

The allele 4.5R of the VNTR. The 4.5R refers to an allele with a length between 4 and 5 repeats. Sequencing of this size allele revealed an
insertion of an 18-bp sequence, completely identical with the sequence immediate 30 downstream sequence of the VNTR, to the most common 4R
haplotype.

Table 3 Motif compositions
haplotypes (chimpanzee)

Chi_seq1
Chi_seq2
Chi_seq3
Totalof

the 48-bp

VNTR

N

Motif Compositiona

6
6
8
20

C1 – C3 – C4 – C5 – C6
C1 – C3 – C4 – C5 – C7b
C2 – C3 – C4 – C5 – C7

C, chimpanzee motif; VNTR, variable number of tandem repeat.
A haplotype (C2 – C3 – C8 – C9 – C7) was earlier reported but not
identified in this study.
a
C1 – C9 represent motifs. See Table 2 for detailed information on
motif sequences.
b
Novel haplotype identified in this study.

should have lost the third motif during its evolution.
When we examined chimpanzee haplotypes, order ofmotifs were concordant with corresponding human
motifs: C1 and C2 for H1, C3 – C5 for H2, and C6 and C7
for H4 (see Table 3).
With the assumption of ‘H1 – H2 – H3 – H4’ being theancestral haplotype, we constructed a genetic relationshipof haplotypes of the human VNTR (Figure 3a). Derivation
European Journal of Human Genetics

of most haplotypes can be accounted for by unequalcrossing-over. For example, haplotypes H1 – H4, H1 – H2 –H4, H1 – H2 – H31, H1 – H11 – H4, and H1 – H2 – H3 – H2 – H3 –H4 can be generated from various kinds of unequalcrossing-overs of the most frequent haplotype, H1 – H2 –H3 – H4 (Figure 3a). We have to assume a total of two
nucleotide substitutions (shown with open triangles in
Figure 3a): one from haplotype H1 – H2 – H6 – H5 – H2 – H4 to
H1 – H2 – H6 – H5 – H2 – H20 and another from H1 – H2 – H6 –H5 – H2 – H5 – H4 to H1 – H2 – H27 – H5 – H2 – H5 – H4. Eventhe complex transformation from haplotype H1 – H2 –H3 – H4 to H1 – H2 – H6 – H5 – H2 – H5 – H4 can be reconstructed as six cycles of unequal crossing-overs (Figure 3b).

Association with schizophrenia
In the analysis of 570 cases and 570 controls, no significant
association was found in the global distribution of the
48-bp VNTR size alleles (P ¼ 0.166). (Table 1). Regarding
individual haplotypes, the allele 3R, frequency of which
was only 0.003 and 0.012 in cases and controls, respectively, was significant (P ¼ 0.012) by Fisher’s exact test. The
comparison between the allele 7R and all the other allelesin the case-control samples gave an insignificant result

DRD4: selection and association with schizophrenia
E Hattori et al

797

Table 4

Haplotypes of the 48-bp VNTR and adjacent two SNPs (human)

AlleleN

Motif compositiona

rs1870723b

rs7482904b

2R

4
1
3
2
12
1
3
3
1
1
3
2
36

H1 – H4
H1 – H4
H1 – H2 – H4
H1 – H2 – H31
H1 – H2 – H3 – H4
H1 – H2 – H13 – H4
H1 – H2 – (18-bp) – H3 – H4
H1 – H11 – H2 – H3 – H4
H1 – H2 – H6 – H5 – H2 – H20
H1 – H2 – H6 – H5 – H2 – H4
H1 – H2 – H6 – H5 – H2 – H5 – H4
H1 – H2 – H13 – H5 – H2 – H5 – H4

A
G
G
G
G
A
G
G
G
G
G
G

C
G
G
G
G
C
G
G
C
C
C
C

3R
4R
4.5Rc
5R
6R
7R
Total

VNTR, variable number of tandem repeat.
aNumbers represent motifs. See Table 2 for detailed information on motif sequences.
b
SNPs rs1870723 and rs7482904 are 165-bp and 185-bp downstream of the VNTR, respectively.
c
The 4.5R refers to an allele with a length between 4 and 5 repeats. See Figure 1 for details.

Discussion

H1

C1

H3
1
2*,31*

27,33,37

31*

C2

11
11

11

21,35*,38
H4

C9
45

16,24,25
46

35*
4,30

12,33

C6

C4

44
C8

H2
4,30

14
43,47

44

C7
C3

2*
C5

Figure 2

Phylogenetic network for human and chimpanzee motifs.
Motifs H1 – H4 and C1 – C9 are from human and chimpanzee samples,
respectively. See Table 2 and Supplementary Table S1 for detailed
information on motif sequences. Black circles designate possible nodesof common ancestor for human and chimpanzee lineages. Numbers
on lines are nucleotide site positions, and those with asterisks indicate
parallel changes. C1 and C2 differ by only one nucleotide, and they areclosest to H1. C3, C4, C5, C8, and C9 are similar with each other, andthey are closest to H2. Remaining two chimpanzee motifs (C6 and C7)
differ by only one nucleotide, and they are closest to H4. There is no
chimpanzee motif that is close to H3.

(P ¼ 0.163) (Table 1). Evidence of association was not foundin the analysis of 124 pedigrees with schizophrenic
offspring (global P ¼ 0.506). None of the individual alleles
were significant and the allele 7R was not observed in thispedigree data set (Table 5).

In this study of DRD4, we particularly focused on the VNTR
in exon 3 in terms of documented evidence of selective
sweep acting on the allele 7R of the VNTR. It is proposedthat the allele 7R was originated as a rare event involving
both mutations and gene conversions and prevailed
rapidly by positive selection.34,35 However, the allele 7R
was found very rare (0.5%) in our Japanese samples, andthis agreed with an earlier report.31 – 33 This low frequencyof the allele 7R was also observed in Chinese populations.20,26 Also, the allele 7R of an African population
was reported to have an intermediate frequency of 0.21.44
This implies lack of selective sweep at this locus at least in
Asian population history. Therefore, we further studied theentire VNTR region by re-sequencing both the Japanesehuman and chimpanzee samples.
The most common 4R (H1 – H2 – H3 – H4) was considered
ancestral haplotype, as three of the constituent motifs were
found connected to chimpanzee motif clusters andthe order of motifs of this haplotype is consistent withthat of chimpanzee. Although 4R (H1 – H2 – H3 – H4) was
postulated to be ancestral haplotype earlier based on thehuman motif frequencies,35 the current study is first to
provide support for this inference by phylogenetic analysis
program exploiting both human and chimpanzee
sequence data.
A two-SNP haplotype ‘rs1870723G – 7482904G’, which isin synteny with the most common 4R (H1 – H2 – H3 – H4)
VNTR, is considered to constitute the distal part of theancestral haplotype. This two-SNP haplotype alongwith ‘rs1870723A – 7482904C’ have been reported,but the intermediate ones (‘rs1870723G – 7482904C’ and‘rs1870723A – 7482904G’) were missing in the earlier
studies. This study detected an intermediate haplotype‘rs1870723G – 7482904C’ attached to multiple VNTR haplotypes. For this G – C haplotype to be present, either aEuropean Journal of Human Genetics

DRD4: selection and association with schizophrenia
E Hattori et al798
1–2–31
1–2–4
1–2–3–4
1–2–3–4

1–11–4**

1–2–3–4 1–2–3–4
1–2–3–4 1–2–3–4

1–11–4
1–2–3–4

1–11–2–3–4

1–4

1–2–3–4
1–2–3–4
1–2–3–4

1–2–3–4
1–2–3–4

1–2–3–2–3–4*
1–2–3–4
1–2–6–5–2–5–4

1–2–6–5–2–5–4
1–2–3–4

1–2–6–5–2–4

1–2–13–4

1–2–6–5–2–20

1–2–6–5–2–5–4

1–2–13–4
1–2–6–5–2–5–4

1–2–27–5–2–5–4

1–2–13–5–2–5–4

4R

1

2

2

3

4

2R

1

4

1

2

3

4

2R

1

3R

1

4R

4R

4R

4R

1

3

4
4R

4R

1

2

3

1

2

3

4

3R

1

2

20

3R

1

2

20

4

4R

1

2

3

4

5

4

4R

1

2

6

4

1

2

3

4

4R

1

2

6

4

3R

1

5

4

4R

1

2

3

4

1

2

5

4

6

5

2

3

4

6R

1

2

6

2

3

4

4R

1

2

5

4

6

5

2

5

4

7R

7R

1

1

2

2

6

6

7R

1

2

5

2

3

4

3R

1

5

4

5

2

5

4

6 cycles unequal crossing-overs

3R

4

7R

6R

1

2

1

1

2

20

4R

1

2

3

4

2

6

2

3

4

6 cycles unequal crossing-overs

Figure 3 A possible genetic relationship of haplotypes of the human VNTR. (a) Possible transformation from haplotype 1 – 2 – 3 – 4 to 1 – 2 – 6 – 5 –2 – 5 – 4. Unequal crossing-over events and nucleotide substitutions are indicated by black circles and open triangles, respectively. Arrows indicate
direction of changes. Estimated patterns of unequal crossing-over are shown next to product of crossing-overs. The asterisks point to recombination
within motifs, which give rise to new motifs. (b) Presumed pathways of how the 7R allele can be generated from the 4R allele with possible
intermediate haplotypes. The prefix ‘H’ for each motif is omitted.

nucleotide substitution or a recombination in the very
short genomic interval between these two sites (20-bp
long) must occur.
European Journal of Human Genetics

From the VNTR haplotype data, the most frequent 7R(H1 – H2 – H6 – H5 – H2 – H5 – H4) haplotype can be generated
by only six unequal crossing-overs. Five descendentDRD4: selection and association with schizophrenia
E Hattori et al

799

Table 5 Transmission disequilibrium test for the VNTR in
124 pedigrees
AlleleFrequency

P*

2R
3R
4R
5R
6R

0.091
0.012
0.852
0.040
0.005

0.275
0.317
0.602
0.782
0.564
Global P ¼ 0.506**

Abbreviation: VNTR, variable number of tandem repeat.
*P-value for individual alleles was calculated by FBAT program.
**Global P-value was calculated by PDT program.

haplotypes (1 – 2 – 13 – 5 – 2 – 5 – 4, 1 – 2 – 13 – 4, 1 – 2 – 27 – 5 –2 – 5 – 4, 1 – 2 – 6 – 5 – 2 – 4, and 1 – 2 – 6 – 5 – 2 – 20 in Figure 3)
were thought to have been derived from this 7R haplotype.
Our sequence data on rs1870723 and rs7482904 revealed
the prevalence of the intermediate two-SNP haplotype
attached exclusively to this common 7R and its descendants. Thus the lineage downstream of the most frequent
7R (H1 – H2 – H6 – H5 – H2 – H5 – H4) is thought to be longenough to add diversity to the sequence around the VNTR.
Thus our findings do not agree with earlier documentedlines of evidence for selection favoring for the allele 7R,
which claim complexity of its generation involving multiple mutations and gene conversions and its high prevalence despite short lineage.35 We, therefore, argue that the
drastic difference in the frequency of the allele 7R among
populations may well be a consequence of random genetic
drift. The possibility of balancing selection or different
selective forces acting on different populations cannot
completely be ruled out. In the case of the angiotensinogengene (AGT) polymorphism, this idea was supported by
supposedly varying degree of physical advantages of waterretention in different geographical conditions.44 It may
be, however, difficult to formulate population-specificadvantages for a specific psychiatric trait that the DRD4
may affect.
We further investigated this region for association withschizophrenia. No evidence of global association was
found between the 48-bp alleles of VNTR and the disease.
The allele 3R showed significant under-representation incases than in controls. This allele, however, was observedin only 14 chromosomes (1.2%) in controls. The possibilityof the allele 3R being true protective variant for only asmall fraction of subjects cannot be excluded, although
this apparent association of a rare allele may be because of
population stratification. Association of DRD4 VNTR withschizophrenia has not been consistently shown in theearlier studies. Although a role of this polymorphism infemale schizophrenia was not be excluded by a metaanalysis,45 our data provided no evidence of genderspecific association. Earlier, we suggested a possible roleof promoter variants of this gene in the development ofschizophrenia.46 Promoter variant and VNTR size alleles
are in LD both in our Japanese samples (data not shown)and in world-wide populations.34 Documented associations of the VNTR with ADHD and with other psychiatrictraits may reflect LD between VNTR alleles and promoter
variants.
There are limitations in this study. First, as DRD4 VNTR isa complex repeat polymorphism involving historical
recombinations and was not subject to rigorous statisticaltests for selection, although this problem is shared by all
studies on the same subject. Second, our association
analysis is based on a case-control design, where both
false-positive and false-negative results can be produced.
Though our analysis of the same sample set using
STRUCTURE software47 detected no evidence of population stratification.38 Third, the sample size in the association study is obviously limited. When the genotypic
relative risk is set to 1.2 with the multiplicative model,
the current size of case-control samples had a power ofdetecting 0.313 and 0.360 at the threshold of P ¼ 0.05 evenfor common size alleles 2R and 4R, respectively.49 Conversely, to gain the power of 0.8 for detecting a risk allele
with the same relative risk, the sample size would need tobe 2100 cases and 2100 controls for the allele 2R, and 1700
cases and the 1700 controls for the allele 4R.48 Fourth, inassociation analysis, alleles were coded according to theirlengths. This introduces loss of information on sequence
diversity, because even the same size allele is known tohave multiple variants according to its constituent motifs.
This sample size would not have power to detect association of each VNTR haplotype, many of which have low
frequencies.

Acknowledgements
We are grateful to all the patients and their families who participatedin this study. This study was supported by RIKEN BSI Funds, Grant-inaid for scientific studies from the MEXT of Japan to NS and EH,
NARSAD Young Investigator Award to EH, and CREST funds from theJapan Science and Technology Agency, Japan to TY. We wish to thankthe members of the Research Resource Center at the RIKEN Brain
Science Institute for sequencing and genotyping services.

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Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)

European Journal of Human Genetics