Haplotype diversity and linkage disequilibrium at the DRD2 locus among the tribes of western and southern regions of India

BACKGROUND: Dopamine receptor D2 (DRD2) is an important gene having functional significance in the fields of neuropsychiatry and pharmacology and also has importance in evolutionary studies. MATERIALS AND METHODS: This study was undertaken to find out the haplotype distribution and linkage disequilibrium (LD) pattern for the three TaqI sites (TaqI ‘A’, TaqI ‘B’ and TaqI ‘D’) in the DRD2 gene in 232 unrelated individuals from five ethno-linguistically distinct endogamous tribal populations; Siddis and Gonds of Uttara Kannada district, Karnataka; Varli and Kolgha of Valsad district, Gujarat; and Dangi Konkana of Dang district, Gujarat. The genotype data obtained after molecular analysis of the three DRD2 sites was subjected to statistical analysis such as calculation of allele frequencies, haplotype frequencies among others. Subsequently, a neighbor-joining tree was also constructed from the data obtained. RESULTS: The three DRD2 sites were found to be polymorphic in all the populations. All the populations showed high levels of heterozygosities. Out of the eight possible haplotypes, most populations shared seven haplotypes. Of all the populations, Siddis showed the highest frequency of the ancestral haplotype B2D2A1 (11.4%). Significant LD was found to exist for TaqI ‘A’ and TaqI ‘B’ sites in both the populations. CONCLUSION: The findings are in concurrence with those from other Indian studies, especially from Dravidian-speaking South Indian populations. Similar pattern of diversity observed for ethnically and linguistically diverse populations in the present study is indicative of complex structure of Indian populations.


Introduction
Over the years, population genetic studies have seen Kannada district is primarily distributed in six talukas, namely, Sirsi, Mundagoda, Joida, Haliyala, Yellapura, and Ankola. [6] In their somatoscopic features (broad nose, dark skin color, kinky hair, alveolar prognathism, etc.), the Siddis resemble Africans even today. The Siddis also show sign of the clan system that once existed among the Siddis of Africa. [6] Ethno-historical records have revealed a long period of Portuguese and South Indian contact with the Siddis, and therefore, biological affinities of the Siddis with them cannot be ruled out. The social and cultural ties of the Siddis to their immediate neighbors are so strong that they have become bilingual and speak Konkani, a language belonging to the Indo-European family of languages, and Kannada, a language belonging to the Dravidian group of languages, and have abandoned the Swahili family of languages to which they originally belonged. Each of the four divisions has exogamous clans. Their population size in Gujarat is 255, 271. [7] Linguistically, they are affiliated to Indo-European language family.  [7] Kolgha: The Kolghas are classified as a primitive tribal group (PTG) in Gujarat state of India and their population size is 48,000. [7] Spoken dialect of Kolgha has a strong admixture of Indo-European and Dravidian language family words. They are mainly dependent on labor, cattle grazing and tanning of animal hides for their subsistence.
Kolghas are divided into several exogamous clans.
The gene under study, DRD2, spans over 270 kb and has been mapped to locus 11q22.3-q23.1. [8] It encodes the D2 subtype of dopamine receptor which is one of the five types of dopamine receptors encoded by five separate genes. These receptors are known to mediate enzyme activities, metabolic rates, and ion channels and are involved in neurological signaling and functioning. [9] DRD2 gene is of special interest as it is a target site of many neuropsychiatric drugs, and is thus of prime concern in the fields of neurology, psychiatry, endocrinology, among others. It is a strong candidate gene implicated in alcoholism and other substance use disorders. [10][11][12] Beginning with detection of TaqI 'A' site, [13] several other restriction polymorphism sites have been identified mostly in the non-coding region of this gene, [14] of which the TaqI 'A' site is the one most frequently studied in association studies. [13,15] The gene is being increasingly studied because of not only its functional significance but also its evolutionary significance. Three restriction site polymorphisms (RSPs) that are of special interest in finding out evolutionary relationships with reference to the DRD2 locus are TaqI  B2, D2 and A1 alleles are the ancestral alleles. [2,15,16] A total of 232 chromosomes were typed in the five populations for the three autosomal co-dominant biallelic  Table 1.
The three sites, TaqI 'A', [17] TaqI 'B' [18] TaqI 'A,' [19] have been described previously. Five milliliters of intravenous blood was collected from individuals unrelated up to at least first cousin level by a trained medical practitioner after taking informed consent from them. Following blood collection, DNA was isolated using salting-out method. [20] The three DRD2 sites were amplified using the standard primers and protocols. [15,21] The polymerase chain reaction (PCR) products were then digested with the restriction enzyme TaqI as per the manufacturer's recommended conditions. Electrophoresis was subsequently carried out in 2% agarose gel stained with ethidium bromide for visualization. Following this, data obtained were subjected to statistical analysis.
All the procedures of data collection and analysis were in accordance with ethical standards of the Helsinki Declaration (1975).

Results
Allele frequency estimates for the three DRD2 sites in the five populations were made by direct gene counting and the assumption of Hardy-Weinberg Equilibrium was tested using χ 2 goodness-of-fit test. The estimates are presented in Table 2. Average heterozygosity was computed according to Nei [22] and the values are presented in Table 3. Within each population, haplotype frequencies were estimated by maximum likelihood method from the multisite marker typing data, using the program HAPLOPOP.
The haplotype frequencies for the five populations are presented in Table 4.   The ancestral haplotype B2D2A1 is found to be present in highest frequency in Siddis (11.4%) and lowest in Dangi The standardized pairwise linkage disequilibrium (LD) value D′ was computed for each pair of markers [23] Data on pairwise LD values for the three DRD2 sites are shown in Table 5. A dendrogram was constructed using the neighborjoining (NJ) method [24] to identify affinities among the study populations and is given in Figure 1.
Siddi population is grouping with Gonds on one hand and Kolgha is lying on the other extreme of the NJ tree and is closer to Dangi Konkana and Varli than the two South Indian populations.

Discussion
The three TaqI sites define evolutionary relationships of the DRD2 haplotypes. Alleles at the three sites show a variable pattern of distribution across different populations of the world or more so across different geographic areas. Ancestral allele B2 is observed at higher frequencies than B1 allele in African (>0.72) and European populations (>0.68) as compared to other world populations. [2,25,26] Ancestral allele D2 has higher frequency than D1 in all the world populations with the    ancestral allele A1 is found to have lower frequency than A2 in all world populations except in American populations. [2] In the Indian context it is seen that the frequency of ancestral allele B2 at TaqI 'B' site varies from 36.67% in Onge tribe [27] to 91% in Toda tribe. [25] It is observed that range is not a reliable parameter  [25,[27][28][29] but not close to the median value of 0.9 and 0.85 obtained for African and European populations, respectively, [2] and the median value of 0.81 computed for Indo-European speaking North Indian population groups. [30]  Indian studies. [25,[27][28][29] As also seen in other studies, [25,27,28] [2] that the ancestral haplotype is common only in Africa but is rare or absent elsewhere. Siddi especially is showing the ancestral haplotype in appreciable frequency (11.4%). This frequency is higher than that observed in most Indian studies, [25,28] but lower than that found for Siddis of Gujarat [27] and Thotis and Nayakpods of Andhra Pradesh. [29] The values observed are comparable to findings from other South Indian studies [25,[27][28][29]  Also, Kolgha, a PTG, is positioned separately from other groups but is closer to its geographic neighbors. Although only one locus has been considered in the present study, haplotype analysis is known to be a robust method for studying population structure. Thus, the results obtained in the present study indicate that the structure of Indian populations is complex and is the by product of cultural, temporal and spatial changes over a period of time.