Neurol Sci (2009) 30:487–493
DOI 10.1007/s10072-009-0139-2
ORIGINAL ARTICLE
Dopamine transporter (DAT1) VNTR polymorphism
in 12 Indian populations
L. V. K. S. Bhaskar Æ Kumarasamy Thangaraj Æ Connie J. Mulligan Æ
Samiksha Wasnik Æ Amrita Nandan Æ Varun Kumar Sharma Æ Vishwas Sharma Æ
Alla Govardhana Reddy Æ Lalji Singh Æ Vadlamudi Raghavendra Rao
Received: 21 August 2008 / Accepted: 31 August 2009 / Published online: 25 September 2009
Ó Springer-Verlag 2009
Abstract The dopamine transporter (DAT1) is a mem-
brane spanning protein that binds the neurotransmitter
dopamine and performs re-uptake of dopamine from the
synapse into a neuron. The gene encoding DAT1 consists
of 15 exons spanning 60 kb on chromosome 5p15.32.
Several studies have investigated the possible associations
between variants in DAT1 gene and psychiatric disorders.
The present study aimed to determine the distribution of
the variable number of tandem repeat (VNTR) polymor-
phism in the 30 untranslated region of DAT1 in 12 Indian
populations. A total of 471 healthy unrelated individuals in
12 Indian populations from 3 linguistic groups were
included in the present study. The analysis was carried out
using PCR and electrophoresis. Overall, 4 alleles of the
DAT1 40-bp VNTR, ranging from 7 to 11 repeats were
detected. Heterozygosity indices were low and varied from
0.114 to 0.406. The results demonstrate the variability of
the DAT1 40-bp VNTR polymorphism in Indian popula-
tions and revealed a high similarity with East Asian
populations.
L. V. K. S. Bhaskar
K. Thangaraj
S. Wasnik
A. Nandan

V. K. Sharma
V. Sharma
A. G. Reddy
L. Singh
Centre for Cellular and Molecular Biology, Hyderabad, India
C. J. Mulligan
Department of Anthropology, University of Florida,
Gainesville, Florida, USA
V. R. Rao (&)
Anthropological Survey of India, 27-Jawaharlal Nehru Road,
Kolkata 700016, India
e-mail: drraovr@yahoo.com
L. V. K. S. Bhaskar
Department of Biomedical Sciences,
Sri Ramachandra University, Chennai, India
Keywords DAT
VNTR
Allelic variation

Indian populations
Introduction
Variable number of tandem repeats (VNTRs) are highly
repetitive DNA sequences in the human genome. VNTRs
show substantial allelic variability in the number of repeat
units as a consequence of a high mutation rate. The vari-
ability shown by many of the VNTRs studied renders them
interesting for different applications, including linkage
analysis, disease, forensic identification, paternity testing,
anthropological research and phylogenetic studies. Recent
data show that such polymorphisms can be useful for
genetic population studies. Dopamine (DA) is a biogenic
amine and is a key neurotransmitter in brain areas involved
in cardiovascular, renal, hormonal, and central nervous
system regulation. The DA transporter DAT1 mediates
the active re-uptake of DA from the synapse and is a
principal regulator of dopaminergic neurotransmission [1].
The gene encoding DAT1 (gene symbol SLC6A3) consists
of 15 exons spanning 60 kb on chromosome 5p15.32
[MIM*126455] [2]. More than one hundred studies have
assessed possible associations between variants in SLC6A3
and psychiatric disorders [3–5]. The polymorphism is
determined by the number of repeats ranging from 3 to 11
copies (the length of the repeat unit is 40 bp) [2]. In
addition to it numerous studies, both population and
family-based, have investigated the role of DAT in vari-
ous diseases implicating the nigrostriatal dopaminergic
system. Parkinson’s disease is characterized by loss of
dopaminergic neurons in the substantia nigra. Further-
more, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)
induced parkinsonism is dependent on DAT [6]. The
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Table 1 Name of the population, their linguistic affiliation, geographic location and number of samples analyzed
S. No Population Region Linguistic group Number of chromosomes (2N)

1 Lingayat Tamil Nadu Dravidian 72

2 Badaga Tamil Nadu Dravidian 128
3 Kota Tamil Nadu Dravidian 108
4 Muduga Kerala Dravidian 96
5 Kurumba Kerala Dravidian 68
6 Adiyan Kerala Dravidian 84
7 Agnikula Kshtriya Andhra Pradesh Dravidian 76
8 Gandla Andhra Pradesh Dravidian 50
9 Sonr Madhya Pradesh Indo-European 78
10 Rajgond Madhya Pradesh Indo-European 70
11 Ojha Madhya Pradesh Indo-European 50
12 Korku Maharastra Austro-Asiatic 62
Total 942

Fig. 1 Map of India with the

locations of different
populations given in Table 1.
The lines within the map
indicate the borders of different
states

hyper-mutability of VNTR loci along with their relative
abundance in the genome makes them excellent DNA
markers for the study of human genetic polymorphism.
Most of the polymorphic VNTRs are localized to non-
coding regions of the human genome where they are
thought to be selectively neutral. There are only a few
examples of VNTRs localized to coding regions [7]. The 30
DAT1 VNTR polymorphism is not associated with muta-
tions in the DAT protein sequence [8]. Population differ-
ences in the DAT1 allele frequency distribution patterns

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Table 2 Allele frequencies and Hardy–Weinberg proportions of the non-tribes. The 532 tribal communities, who are assumed to
DAT1 VNTR polymorphism in 12 Indian populations be the aboriginal inhabitants of the sub-continent, constitute
Allele frequency HW v2 P value 7.76% of the total population (http://www.censusindia.net).
A few studies have reported closer affinity of Indian castes
7 9 10 11
with either European or Asian populations [11]. Our pre-
Lingayat 0.027 0.250 0.722 7.177 0.066 vious study suggested that the vast majority of the Indian
Badaga 0.211 0.734 0.055 4.981 0.173 maternal gene pool ([98%), consisting of Indio-European
Kota 0.241 0.759 0.009 0.923 and Dravidian speakers, is genetically more or less uniform
Muduvan 0.073 0.845 0.063 6.622 0.849 [12]. Blood samples were collected from adult individuals
Kurumba 0.132 0.868 0.791 0.374 belonging to 12 Indian ethnic populations chosen to
Adiyan 0.012 0.131 0.845 0.012 10.371 0.110 encompass the full range of Indian social structure. About
Agnikula Kshatriya 0.079 0.908 0.013 2.935 0.399 5 ml of intravenous blood was collected from 471 indi-
Gandla 0.020 0.140 0.840 7.370 0.061 viduals in EDTA vacutainers. All individuals were normal
Sonr 0.013 0.115 0.859 0.013 8.336 0.215 and healthy volunteers, and no diagnosis for any disease
Rajgond 0.057 0.943 0.129 0.720 was performed. Informed written consent was obtained
Ojha 0.020 0.240 0.740 6.932 0.074 from each individual at the time of blood collection. DNA
Korku 0.016 0.177 0.806 6.800 0.079 from all the samples was isolated using a published protocol
Fst 0.013 0.021 0.019 0.021 [13]. Genotypic analysis of the dopamine transporter gene
polymorphism was performed using the polymerase chain
reaction with the following oligonucleotide primers: 50 -T
have been described [9, 10]. The genomic architecture of GTGGTGTAGGGAACGGCCTGAG-30 and 50 -CTTCCTG
VNTR polymorphism in natural populations is important to GAGGTCACGGCTCAAGG-30 . After initial denaturation
further our understanding of the human genome and its (5 min at 95°C), 35 cycles of amplification were carried out
complex functions. In this study, we analyzed the allele according to the following scheme: 30 s at 61°C for primer
frequencies of the DAT1 40-bp VNTR locus in normal annealing; 1.5 min at 72°C for DNA synthesis; and 30 s at
individuals from 12 different populations from 3 linguistic 95°C for denaturation. For final elongation, the probes were
groups (eight Dravidian, three Indo-European, and one incubated for 7 min at 72°C and then cooled. The amplified
Austro-Asiatic) living in the Indian sub-continent. products were resolved on 2% agarose gel, and visualized
and documented under UV light using Syngene gel doc
system. The sizes of the amplicons were noted by com-
Materials and methods paring against a 100 bp ladder (NEB). Calculations of allele
frequencies observed and expected heterozygosity levels
India is the homeland for 4,635 well-defined endogamous and F statistics were calculated using the FSTAT program
populations, which are culturally stratified as tribes and [14]. Population genetic structure was then investigated by

Table 3 Genotype frequencies and observed heterozygosities of the DAT1 VNTR in 12 Indian populations
Population Genotype frequency Observed
heterozygosity
7/7 7/9 9/9 9/10 10/10 10/11 11/11

Lingayat 0.056 0.083 0.278 0.583 0.333

Badaga 0.047 0.328 0.531 0.078 0.016 0.406
Kota 0.056 0.370 0.574 0.370
Muduvan 0.021 0.104 0.771 0.083 0.021 0.187
Kurumba 0.265 0.735 0.265
Adiyan 0.024 0.048 0.143 0.762 0.024 0.191
Agnikula Kshatriya 0.026 0.105 0.842 0.026 0.132
Gandla 0.040 0.120 0.080 0.760 0.080
Sonr 0.026 0.205 0.744 0.026 0.256
Rajgond 0.114 0.886 0.114
Ojha 0.120 0.200 0.680 0.320
Korku 0.032 0.065 0.194 0.710 0.226

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490
Fig. 2 DAT1 40 bp allele frequency distribution in various world
populations. 1 Biaka; 2 Biaka; 3 Yoruba; 4 Yoruba; 5 Ibo; 6 Hausa; 7
Mbuti; 8 Jews; Ethiopian; 9 AfricanAmericans; 10 Druze; 11 Jews,
Yemenite; 12 Iranian; 13 Arab; 14 Gilaki; 15 Lur; 16 Mazandarani;
17 Easfahan; 18 Kerman; 19 Mashaad; 20 Yazd; 21 Shiraz; 22
Bandari; 23 Azeri; 24 Kurdish; 25 Sistani and Balouchi; 26 Bashkirs;
27 Tatars; 28 Udmurts; 29 Chuvashes; 30 Maris; 31 Mordovians; 32
Komis; 33 Russians; 34 Adygei; 35 Danes; 36 Samaritans; 37 Irish;
an analysis of variance (ANOVA) among nine population
groups representing different broad continental areas
(Africa, West Asia, Central Asia, Europe, South Asia, East
Asia, Oceania, North America, and South America). We
also calculated ANOVA to investigate the relationship
between genetic diversity and the geographical region and
linguistic groups of the study populations.
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Neurol Sci (2009) 30:487–493
38 Finns; 39 Russians; 40 French; 41 Europeans, Mixed; 42 Lingayat;
43 Badaga; 44 Kota; 45 Muduvan; 46 Kurumba; 47 Adiyan; 48 Agni
Kula Kshatriya; 49 Gandla; 50 Sonr; 51 Rajgond; 52 Ojha; 53 Korku;
54 Han; 55 Han; 56 Ami; 57 Japanese; 58 Hakka; 59 Atayal; 60
Cambodians, Khmer; 61 Han; 62 Atayal; 63 Ami; 64 Bunun; 65
Paiwan; 66 Yakut; 67 Melanesian, Nasioi; 68 Micronesians; 69 Maya,
Yucatan; 70 Pima, Mexico; 71 Pima, Arizona; 72 Cheyenne; 73
Karitiana; 74 Ticuna; 75 Surui; 76 Southwest Amerindians
Results
The polymorphic 40 bp VNTR was examined by PCR in
12 Indian populations (Table 1; Fig. 1). The distributions
of allele frequencies and genotype frequencies are shown
in Tables 2 and 3, respectively. None of the population
deviated significantly from Hardy–Weinberg equilibrium
Neurol Sci (2009) 30:487–493 491

Table 4 Significance levels (ANOVA P value) of comparison of allele frequencies of present study populations (south Asian) with different
broad continental areas
Allele 3 Allele 6 Allele 7 Allele 8 Allele 9 Allele 10 Allele 11 Allele 12

Africa 0.000* 1.000 0.000* 0.775 0.026* 0.001* 0.109 0.002*

West Asia 0.062 1.000 0.928 0.587 0.000* 0.000* 0.586 1.000
Central Asia 1.000 0.075 0.467 0.276 0.000* 0.000* 0.632 1.000
Europe 1.000 0.312 0.724 0.344 0.001* 0.211 0.168 1.000
East Asia 1.000 1.000 0.695 0.339 0.298 0.059 0.246 1.000
Oceana 1.000 1.000 0.787 0.953 0.341 0.305 0.242 1.000
North America 1.000 1.000 0.720 0.473 0.039* 0.017* 0.212 1.000
South America 1.000 1.000 0.720 0.473 0.021* 0.009* 0.123 1.000
* Statistically significant

(HWE, Table 2). The analysis detected 4 alleles with 7–11
repeats out of 942 chromosomes typed. The allele spectra
in Indian populations are unimodel with a peak at alleles 9–
10. These patterns coincide with the East Asian population
allele frequency profiles for this locus (Fig. 2). The 10-
repeat allele was most frequent in all of the populations
examined. The frequency of this allele varied from 0.722 in
Lingayat to 0.943 in Rajgond. The frequency of the second
most common allele, carrying 9-repeat units, ranged from
0.057 in Rajgond to 0.250 in Lingayat. The 11-repeat allele
was observed in 5 populations with a frequency range from
0.012 (Adiyan) to 0.063 (Muduvan). The 7-repeat allele
was observed in six populations with the frequency range
from 0.012 (Adiyan) to 0.027 (Lingayat). Heterozygosities
range from 0.114 in Rajgond to a high of 0.406 in the
Badaga.
The distribution of these DAT1 allele frequencies among
the ethnic groups was found to be highly homogeneous and
statistically non-significant for both geographical region
and linguistic groups of the present study (Oneway
ANOVA; P [ 0.05) (results not shown). ANOVA results
using the allele frequency distribution patterns among
different broad continental areas are presented in Table 4.
The present studied populations show statistically signifi-
cant differences with African populations for alleles 3, 7, 9,
10 and 12, but allele 9 has not shown any significant dif-
ference only in East Asian and Oceana populations. The
most frequent ten-repeat allele does not deviate signifi-
cantly from Europe, East Asia and Oceana populations.
Discussion
Several studies have examined the relationship of genotype
of the DAT 40 bp VNTR to DAT binding sites in the
brain. A single positron emission computerized tomo-
graphy (SPECT) analysis, using [3H]M-CIT as ligand,
demonstrated a small 13% decrease in binding to the
transporter in the striatum in the 10/10 genotype group
[15]. Miller and Madras [16] reported that the 9-copy allele
showed increased transcription relative to the 10-copy
allele in heterologous cells (HEK-293). Contrary to this
result, Fuke et al. [17] demonstrated that the 10-copy
VNTR increased gene expression compared to the 7 or 9
copy alleles.
In the present study, we analyzed natural variation in the
DAT1 40-bp VNTR locus in 12 Indian populations. As
established previously [8–10, 18, 19], this locus is extre-
mely polymorphic in populations and significant diversity
has been found for allele spectra in groups of different
origin; similar results were observed here. We assayed
DAT1 VNTR diversity in populations belonging to dif-
ferent linguistic groups of India (Indo-European, Dravidian
and Austro-Asiatic). ANOVA showed no significant dif-
ferences in the allele frequencies among populations based
on geographical region and linguistic affiliation, in spite of
their geographical proximity and population history. The
significant differences in the allele frequency distribution
of 3-, 7-, and 12-repeat alleles with African populations
may be due to restricted distribution in African population.
The 10-repeat allele was most common, present at high
frequency (*72 to 94%), followed by the 9-repeat allele.
However, numerous studies have demonstrated that great
differences exist in allele frequencies across different eth-
nic groups [20–22]. Alleles 9 and 10 did not show signif-
icant differences with East Asia and Oceana populations
when compared with the present study populations. Indeed,
some populations of North/Central or South American
ancestry demonstrated only the 10-repeat allele whereas in
some African and Middle Eastern and Iranian populations,
the 10-repeat allele was as low as 0.35–0.50 [18, 22].
A population with Siberian ancestry revealed further het-
erogeneity that included both a relatively high frequency of
the 7-repeat allele and the presence of a 13-repeat allele

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[23]. A population with Pars ethinicity from Mashhad an
Iranian population showed the 8-repeat allele as a dominant
allele with a frequency of 0.652 [22]. The 6-repeat allele
was reported in Udmurts and Komis populations of Volga-
Ural region [19] and some Iranian populations [22].
A series of studies have attempted to establish a rela-
tionship between the DAT 30 VNTR and alcoholism. One
group demonstrated an increased frequency of the 9- versus
10-repeat allele in German alcoholics experiencing with-
drawal seizures and/or delirium compared to controls or
alcoholics negative for these symptoms [24, 25]. The same
group also demonstrated an increase in the 9-repeat allele
in epilepsy, suggesting that the association of this allele
may be with seizure susceptibility rather than alcohol
withdrawal [26]. Other studies failed to establish a role for
the 30 VNTR in alcoholism [27, 28]. A study of Japanese
showed that the 7-, 9-, and 10-repeat alleles had frequen-
cies of 0.013, 0.06 and 0.90, respectively, in controls, while
alcoholics with a mutation in the aldehyde dehydrogenase-
2 gene had a 2.5-fold greater frequency of the 7-repeat
allele [29]. This suggests that different allelic variants may
contribute to disease in populations of different genetic
background. This may reflect linkage with other variants
in the same gene rather than association with the VNTR
per se. Our study adds to the knowledge on the distribution
of DAT1 gene VNTR polymorphism among the Indian
populations.
Acknowledgments This research work was supported by a grant
from Department of Biotechnology, Government of India (project no.
BT/PR3607/SPD/09/261/2003).
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