No Evidence for Strong Recent Positive Selection Favoring the 7 Repeat Allele of VNTR in the DRD4 Gene

Abstract

The human dopamine receptor D4 (DRD4) gene contains a 48-bp variable number of tandem repeat (VNTR) in exon 3, encoding the third intracellular loop of this dopamine receptor. The DRD4 7R allele, which seems to have a single origin, is commonly observed in various human populations and the nucleotide diversity of the DRD4 7R haplotype at the DRD4 locus is reduced compared to the most common DRD4 4R haplotype. Based on these observations, previous studies have hypothesized that positive selection has acted on the DRD4 7R allele. However, the degrees of linkage disequilibrium (LD) of the DRD4 7R allele with single nucleotide polymorphisms (SNPs) outside the DRD4 locus have not been evaluated. In this study, to re-examine the possibility of recent positive selection favoring the DRD4 7R allele, we genotyped HapMap subjects for DRD4 VNTR, and conducted several neutrality tests including long range haplotype test and iHS test based on the extended haplotype homozygosity. Our results indicated that LD of the DRD4 7R allele was not extended compared to SNP alleles with the similar frequency. Thus, we conclude that the DRD4 7R allele has not been subjected to strong recent positive selection.

About the Authors:

Izumi Naka

Affiliation: Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan

Nao Nishida

Affiliations Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan, Research Center for Hepatitis and Immunology, International Medical Center of Japan Konodai Hospital, Ichikawa, Chiba, Japan

Jun Ohashi

* E-mail: [email protected]

Affiliation: Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan

Introduction

The human dopamine receptor D4 (DRD4; MIM 126452) gene, located on chromosome 11p15.5, contains a 48-bp variable number of tandem repeat (VNTR) in exon 3, encoding the third intracellular loop of this dopamine receptor. Ten VNTR alleles with two (2R) to eleven (11R) repeats have been identified so far. Of these, the DRD4 4R and 7R alleles are commonly observed in human populations [1], while the DRD4 7R allele is very rare in East Asians [2]. Of particular interest, the receptor encoded by the DRD4 7R allele has functional properties different from those by the DRD4 2R and 4R alleles. The DRD4 7R protein, compared with DRD4 2R and DRD4 4R, has different binding affinity to clozapine and spiperone [1], and shows a blunted intracellular response to dopamine [3]. In addition, the repeat sequence of the DRD4 7R allele suppresses the expression level of DRD4 compared to the DRD4 2R and 4R alleles [4]. Furthermore, the DRD4 7R allele has been reported to be associated with behavioral and psychiatric phenotypes such as novelty seeking [5], [6] and attention-deficit hyperactivity disorder [7], [8], even though there are conflicting results [9], [10], [11], [12], [13].

The analysis of the DNA sequences of the DRD4 VNTR alleles and their nucleotide variations suggested that the DRD4 7R allele originated from multiple mutational events [14], [15]. Thus, the DRD4 7R allele observed in human populations is considered to have a single origin. Besides the increased frequency of the DRD4 7R allele, it has been reported that the DRD4 7R allele, unlike 4R, is in strong linkage disequilibrium (LD) with the other DRD4 polymorphisms spanning 6.3 kb [16]. Based on these unique features of the DRD4 7R allele compared to the most common DRD4 4R allele, it has been hypothesized that the DRD4 7R allele has been subjected to positive selection [14], [16].

The extended LD has been regarded as a signature of recent positive selection [17], [18], [19]. However, the genomic region of 6.3 kb previously studied [16] seems to be too narrow to evaluate the possibility of recent positive selection favouring the DRD4 7R. After the original work [16], the international HapMap project [20], [21] provided us a map of several million well-defined single nucleotide polymorphisms (SNPs) in the human genome, and the information about the SNP genotypes of subjects from African, European, and Asian populations are freely available in the public domain. The use of the HapMap data allows us to easily evaluate the degrees of LD of the DRD4 VNTR alleles, since no further genotyping for SNPs flanking the DRD4 locus is necessary. In this study, to re-examine whether the DRD4 7R allele has been the target of recent positive selection, we genotyped DNA samples from the HapMap subjects for the DRD4 VNTR, and conducted neutrality tests including the Long-Range Haplotype (LRH) test [18] and iHS test [22], which are based on the extended haplotype homozygosity (EHH).

Results

Allele frequencies of the DRD4 VNTR

We detected seven VNTR alleles with different lengths (2R, 3R, 4R, 5R, 6R, 7R, 8R) in four HapMap populations [20], [21]: YRI (Yoruba in Ibadan, Nigeria), CEU (CEPH Utah residents with ancestry from northern and western Europe), JPT (Japanese in Tokyo, Japan), and CHB (Han Chinese in Beijing, China) (Table 1). The population frequencies of the DRD4 7R allele were relatively high in African (YRI) and European (CEU) populations, while very low in East Asian (JPT and CHB) populations as reported in the previous study [2]. In this study, the DRD4 VNTR alleles with the same repeat length but with the different VNTR sequences were not distinguished.

[Figure omitted. See PDF.]

Table 1. Allele frequencies of the DRD4 VNTR in HapMap populations.

https://doi.org/10.1371/journal.pone.0024410.t001

To investigate whether the observed distribution of allele frequencies at the DRD4 VNTR is deviated from the expectation under neutrality, we performed the Ewens-Watterson homozygosity test [23], where the sum of squared allele frequencies (F) was calculated for the observed data, and the observed F value was compared with the expected ones generated by simulating samples under neutrality. The P-value was calculated as a proportion of the simulated F value identical or smaller than the observed F in the 10,000 samples simulated, which allowed us to examine both the excess and the deficiency of F. The observed F values in the HapMap populations are slightly larger than the expected ones (Table 1). However, no population showed the significant difference from the neutral expectation in two-sided test (i.e., 0.025<P-value <0.975).

Heterozygosity around the DRD4 locus

A recent positive selection alters patterns of genetic variation in the genomic region adjacent to the targeted gene. During fixation of an advantageous mutation, the variation in the surrounding region is expected to be eliminated due to the hitchhiking effect. In actual, a remarkable reduction in heterozygosity around the ABCC11 gene, which has been subjected to recent positive selection, is observed in East Asian populations [24]. To detect signature of recent positive selection, the degree of heterozygosity around the DRD4 locus was evaluated. Figure 1 shows the averaged heterozygosity across SNPs within 25 kb on either side of the focal SNP in a 1.8-Mb genomic region containing the DRD4 locus in three HapMap populations (i.e., YRI, CEU, and JPT+CHB). The degree of heterozygosity at SNPs close to the DRD4 VNTR was not lower than that of the surrounding region in any HapMap population, implying that any derived allele at DRD4 has not rapidly reached a high frequency (e.g., >0.75). In other words, the DRD4 locus has not experienced a strong and recent selective sweep.

[Figure omitted. See PDF.]

Figure 1. The averaged heterozygosity around DRD4 in HapMap populations.

The X axis represents the genomic position of SNP on chromosome 11. The Y axis represents the averaged heterozygosity calculated for SNPs within 25 kb on either side of the focal SNP. The allele frequency data were obtained from the HapMap database, and only SNPs that were analyzed in three HapMap populations (i.e., YRI, CEU, and JPT+CHB) were selected for the calculation. The dashed line indicates the position of the DRD4 VNTR.

https://doi.org/10.1371/journal.pone.0024410.g001

Genetic differentiation between HapMap populations

Since a local positive selection results in high genetic differentiation between two populations when one population is under selection and the other is not, the high differentiation can be regarded as an evidence of local selection operating at the locus or genomic region. Considering higher population frequencies of the DRD4 7R allele in YRI and CEU than JPT and CHB (Table 1), local positive selection against the 7R allele may have acted in YRI and CEU populations. We examined the degree of differentiation, measured with F_st, for YRI and CEU as candidate populations. The F_st values of the DRD4 7R allele between YRI and JPT+CHB and between CEU and JPT+CHB were 0.15 and 0.07, respectively. We compared these values with those of SNPs on chromosome 11. The comparison revealed that 26220 and 30918 SNPs had F_st values larger than the DRD4 7R allele between YRI and JPT+CHB and between CEU and JPT+CHB, respectively (the empirical P-values were 0.17 and 0.20 for YRI and CEU, respectively). The analysis of F_st provided no evidence of local positive selection acting against the DRD4 7R allele.

Structure of LD around the DRD4 locus

LD plots based on |D'| were visualized for YRI and CEU (Figure 2). Although the allele frequency of 7R was lower than 4R, we found that |D'| values from the DRD4 7R allele to SNPs located outside the DRD4 locus were similar to those from the DRD4 4R allele.

[Figure omitted. See PDF.]

Figure 2. LD structure around DRD4 VNTR.

The |D'| values for all pairwise combinations of SNPs spanning 200 kb and DRD4 VNTR allele (7R or 4R) are visualized. The positions of DRD4 7R and 4R alleles are indicated by arrows. Red squares indicate statistically significant LD (LOD >2) between the pair of loci, and darker colors of red indicate higher values of |D'|. Blue squares indicate |D'| of 1 but without statistical significance. White squares indicate |D'| of <1 with no statistically significant evidence of LD. (A) YRI. (B) CEU.

https://doi.org/10.1371/journal.pone.0024410.g002

LRH and iHS tests for DRD4 VNTR 7R allele

We next calculated the EHH values [18] of the DRD4 7R and 4R alleles in YRI and CEU populations (Figure 3). The 7R-bearing chromosomes have longer LD than 4R-bearing chromosomes in YRI, whereas such a tendency was not found in CEU, regardless of much lower frequency of the DRD4 7R allele compared to the DRD4 4R allele. To examine whether the LD from the DRD4 7R allele has been extended by recent positive selection, we used the LRH test [18] based on the relative EHH (REHH) value, in which the other allele (i.e., reference allele) at the same locus serves as an internal control to normalize recombination rate variation [25]. In this study the REHH value for the focal DRD4 VNTR allele was calculated at a distance of 0.25 centimorgans (cM) from the DRD4 VNTR. The EHH value of the 7R-bearing chromosomes in YRI was 4.58 times greater than the EHH of the reference chromosomes on the centromere-distal side and 4.58 times greater on the centromere-proximal side. Thus, the average of REHH values on both sides was 4.58 ( =  [4.58 + 4.58]/2). In CEU, the REHH values of the 7R-bearing chromosomes were 1.55 and 0 on the centromere-distal and centromere-proximal sides, respectively (i.e., the average REHH was 0.78). To calculate the empirical P-values of the DRD4 7R allele, we obtained the empirical distributions of the REHH values for SNPs whose allele frequencies were similar to the DRD4 7R alleles in YRI and CEU populations (Figure 4). In both populations, the observed REHH values of the 7R-bearing chromosomes were not significantly large compared to the other SNPs on the chromosome 11 (i.e., empirical P-values were 0.17 for YRI and 0.90 for CEU, respectively).

[Figure omitted. See PDF.]

Figure 3. EHH values of DRD4 7R and 4R alleles.

Red and blue lines indicate EHH of the 7R-bearing chromosomes and 4R-bearing chromosomes, respectively. The allele frequencies of DRD4 7R and 4R in each population are presented in parentheses. The horizontal axis represents the physical position of SNPs on the chromosome 11. The position of DRD4 VNTR is indicated by an arrow. (A) YRI. (B) CEU.

https://doi.org/10.1371/journal.pone.0024410.g003

[Figure omitted. See PDF.]

Figure 4. Empirical distribution of REHH of SNPs on the chromosome 11.

For each SNP with an allele whose population frequency was similar to the DRD4 7R allele, the average of REHH values on both sides (i.e., centromere-proximal and cetromere-distal) was calculated, and the empirical distributions for SNPs on the chromosome 11 were obtained. The data of SNPs with the average of REHH more than 15 are excluded from the histogram. REHH was calculated at a distance of 0.25 centimorgans (cM) from the core SNP. REHH value of DRD4 7R is indicated by dashed line. (A) YRI. (B) CEU.

https://doi.org/10.1371/journal.pone.0024410.g004

We further performed the iHS test [22]. In the iHS test, unstandardized iHS value was computed for SNPs with minor allele frequency similar to the frequency of the DRD4 7R allele. Large negative unstandardized iHS values indicate that minor-allele-bearing chromosomes have much longer LD than major-allele-bearing chromosomes. The unstandardized iHS values of the 7R-bearing chromosomes in YRI and in CEU were -1.23 and -0.65, respectively. These values were not large negative when compared with the unstandardized iHS values for SNPs on the chromosome 11 (Figure 5). The empirical P-values for the DRD4 7R allele were 0.46 for YRI and 0.60 for CEU, respectively.

[Figure omitted. See PDF.]

Figure 5. Empirical distribution of unstandardized iHS values of SNPs on the chromosome 11.

For each SNP with an allele whose population frequency was similar to the DRD4 7R allele, the unstandardized iHS was calculated and the empirical distributions for SNPs on the chromosome 11 were obtained. The iHH value (i.e., area under the EHH curve) was calculated until EHH reaches 0.05. Unstandardized iHS value of DRD4 7R is indicated by dashed line. (A) YRI. (B) CEU.

https://doi.org/10.1371/journal.pone.0024410.g005

Power simulation

Neither LRH test nor iHS test provided any evidence of longer LD of the 7R-bearing chromosomes. However, selection intensity for the DRD4 7R allele might be too low to be detected. We assessed the power of the LRH test and iHS test by using a computer simulation. Using SelSim program [26], 120 chromosomes bearing 101 SNPs evenly distributed in the region with the size of 1 centimorgan (cM) were simulated, where a derived allele at the 51th SNP was assumed to be subjected to positive selection. The final population frequency of the selected allele was set to be either 0.15 or 0.3, since the present frequencies of the DRD4 7R allele in CEU and YRI are approximately 0.15 and 0.3, respectively.

The results demonstrated that the power of the LRH test [18] was always smaller than that of the iHS test [22] in the present parameter settings (Figure 6). Here, Ns of 50 corresponds to s of 0.005 in a population with the population size of 10,000. Although our simulation indicated that the LRH test reveals very low statistical power for a selected allele with small selection coefficient, the LRH test successfully detected a significant evidence of positive selection acting at the G6PD gene [18]. Since the selection coefficient of the G6PD allele conferring resistance to malaria has been estimated to be more than 0.1 [27], we may say that the LRH test has enough power to detect such a strong recent selection.

[Figure omitted. See PDF.]

Figure 6. Power of LRH and iHS tests.

White and shaded bars indicate the LRT and iHS tests, respectively. The final population frequencies of the selected allele are (A) 0.15 and (B) 0.3.

https://doi.org/10.1371/journal.pone.0024410.g006

The iHS test achieved high statistical power (>0.8) for Ns (N is diploid population size and s is selection coefficient) of 250 when the final population frequencies of the selected allele were 0.15 and 0.3, but showed low power (< 0.3) for Ns of 50. In addition, we should note that both LRH and iHS tests are expected to attain lower power for actual populations such as YRI and CEU than for the model assumed in our simulation because the variance of test statistic for neutral SNPs is larger in the former due to their complicated population history. Thus, statistical power of the present neutrality tests, LRH and iHS, for the HapMap subjects is too low if selection intensity has not been very high.

Discussion

No significant extended LD from the DRD4 7R allele to SNPs outside the DRD4 locus was detected in the present study. Our results seem to be inconsistent with the previous observation that the nucleotide diversity of the DRD4 haplotype bearing polymorphisms spanning 6.3 kb is more reduced in the DRD4 7R allele than the most common DRD4 4R allele [16]. However, it is not surprising that the diversity of the DRD4 7R allele is lower than that of the DRD4 4R allele if the DRD4 7R allele is derived from the DRD4 4R allele [14].

A previous study also showed that non-synonymous substitutions have occurred more frequently than synonymous ones in the human DRD4 VNTR [14]. This implies that diversifying selection has operated against DRD4 VNTR especially in human lineage, though the length polymorphism at DRD4 VNTR has been commonly found even in nonhuman primate species [28]. However, it is unlikely that the observed bias towards nonsynonymous amino-acid changes has been achieved only by positive selection favoring the DRD4 7R allele because the bias can be found not only in VNTR motifs of the DRD4 7R allele but also in those of the other alleles [14].

For selectively neutral alleles with the similar population frequencies, their age is thought to be inversely proportional to the REHH value. It is not feasible to estimate the age of the DRD4 7R allele based only on the present DRD4 VNTR allele frequency data. However, the observed REHH and unstandardized iHS values for YRI and CEU (Figures 4 and 5) indicate that the age of the DRD4 7R allele is not extraordinarily young compared to SNP alleles with the similar frequencies. Hattori et al. [15] pointed out the possibility that five distinct DRD4 VNTR alleles had been derived from the DRD4 7R allele based on the sequencing analysis of the VNTR region. If this prediction is true, the original DRD4 7R allele is considered to have appeared long time ago. These observations lead us to conclude that the DRD4 7R allele has not been subjected to strong recent positive selection and the increased population frequencies of the DRD4 7R allele in African and European populations would have been caused by random genetic drift, though the possibility that diversifying selection has acted against the DRD4 VNTR in primate species including humans for a long evolutionary time cannot be excluded.

Materials and Methods

Samples

The genomic DNA samples used by the International HapMap Project were obtained from the Coriell Cell Repository: 56 subjects from YRI (Yoruba in Ibadan, Nigeria), 55 CEU (CEPH Utah residents with ancestry from northern and western Europe), 39 JPT (Japanese in Tokyo, Japan), and 44 CHB (Han Chinese in Beijing, China).

Genotyping

For genotyping the VNTR in exon 3 of the DRD4 gene, PCR was performed using the following pair of primers previously reported [29]: upstream 5′-ACTACGTGGTCTACTCGTCCGTGT-3′ and downstream 5′-TCAGGACAGGAACCCACCGA-3′. PCR was performed with an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 30 s, and a final extension at 72°C for 7 min using a thermal cycler (GeneAmp PCR system 9700; Perkin-Elmer Applied Biosystems). The PCR-amplified DNA fragments were analyzed using a microfluidics-based platform (Agilent 2100 bioanalyzer; Agilent Technologies) for accurate sizing with the Agilent 2100 Bioanalyzer DNA 1000 kit (Agilent Technologies).

Statistical analyses

Using Arlequin version 3.5 [30], the Ewens-Watterson test [23] based on Ewens sampling theory of neutral alleles [31] was performed to test whether the observed distribution of allele frequencies at the DRD4 VNTR is deviated from the expectation under neutrality.

The allele frequency data of SNPs on the chromosome 11, analyzed in all the HapMap populations, were obtained from the HapMap database (http://hapmap.ncbi.nlm.nih.gov/index.html.en) and used for the calculation of the heterozygosity. The heterozygosity was calculated for each SNP and then averaged across SNPs within 25 kb on either side of the focal SNP in three HapMap populations (i.e., YRI, CEU, and JPT+CHB).

A pairwise F_st value for each SNP on the chromosome 11 was calculated between HapMap populations based on the following formula [32]: F_st  =  (H_A+B – [H_A+H_B]/2)/H_A+B, where H_A  = 2p_A(1-p_A), H_B  = 2p_B(1-p_B), and  = 2([p_A+p_B]/2)(1-[p_A+p_B]/2). Here, p_A and p_B represent the population frequencies of one of alleles for each SNP in populations A and B, respectively. For the F_st value of the DRD4 7R allele, p_A and p_B are the population frequencies of the DRD4 7R allele in populations A and B.

To evaluate the structure of LD around the DRD4 VNTR, the SNP genotype data of HapMap subjects were obtained from the HapMap database, and the absolute D' values, |D'|, for all pairwise combinations of SNPs with minor allele frequency of more than 0.1 and DRD4 VNTR allele were estimated and visualized using Haploview software [33]. Here, SNPs spanning 200 kb were analyzed. As Haploview program is unable to analyze multi-allelic locus such as VNTR, only one of VNTR alleles was focused and the other VNTR alleles were regarded as the same ones. For example, the DRD4 7R allele and the other VNTR alleles were designated as “A” and “G”, respectively for Haploview analysis.

The phased haplotypes or diplotype in an individual for the calculation of EHH [18] were estimated by using fastPHASE program version 1.2.0 [34]. Like Haploview analysis, in fastPHASE analysis, only one of VNTR alleles was focused and the other VNTR alleles were regarded as the same ones. To conduct the LRH test [18], the empirical distributions of the REHH of SNPs on the chromosome 11 were obtained for YRI and CEU. Only SNPs with similar allele frequency of the DRD4 7R allele were considered. The phased haplotype data were retrieved from the HapMap database. The REHH value was defined as the ratio of the EHH on the tested allele compared with the EHH of the reference allele (i.e., the other allele) at a distance of 0.25 cM from the core SNP. When the EHH of the reference allele was 0, the REHH value was excluded from the empirical distribution.

To perform the iHS test [22], in plots of EHH versus distance, the area under the EHH curve was calculated until EHH reaches 0.05. This integrated EHH (iHH) (summed over both directions away from the core SNP) was computed for each allele of SNPs with minor allele frequency similar to the frequency of the DRD4 7R allele, and was denoted iHH_major for major allele or iHH_minor for minor allele. In this study, test static iHS was defined as follows:

Large negative values therefore indicate long haplotypes carrying the minor allele. Unlike the original study [22], we did not consider the ancestral status of each SNP allele because the ancestral state of each DRD4 VNTR allele is unknown.

Computer simulation for power calculation

Using SelSim program [26] based on the coalescent theory, 120 chromosomes bearing 101 SNPs evenly distributed in the region with the size of 1 cM were simulated assuming a population with constant size, N, of 5000 individuals. In the simulation, a derived allele at the 51th SNP was positively selected with selection coefficient of s, where the dominance parameter was set to 0.5 (i.e., genic selection). It should be noted that the product of N and s (i.e., Ns) is important rather than each parameter in this coalescent simulation. For 120 chromosomes, the EHH values of the derived and ancestral alleles at the 51th SNP for both directions were computed. REHH value at a distance of 0.25 cM from the selected SNP and iHH value were calculated for LRT test [18] and iHS test [22], respectively. We performed 200 simulation runs for deterministic models with s of 0.01 (i.e., Ns of 50) and 0.05 (i.e., Ns of 250). In addition, 1000 runs for neutral model (s = 0) were performed to have the null distributions of the average REHH and unstandardized iHS values. In the calculation of the statistical power, “positive selection” was regarded to be successfully detected in a simulation run when the test statistic (average REHH in LRH test or unstandardized iHS in iHS test) in selection model exceeded the 95th percentile of those observed in neutral model. The proportion of the detection in 200 runs was defined as the statistical power.

Acknowledgments

The authors thank Dr Tsuchiya for his helpful comments and suggestions.

Author Contributions

Conceived and designed the experiments: IN JO. Performed the experiments: IN NN. Analyzed the data: IN JO. Contributed reagents/materials/analysis tools: NN JO. Wrote the paper: IN JO. Performed the computer simulation: JO.

Citation: Naka I, Nishida N, Ohashi J (2011) No Evidence for Strong Recent Positive Selection Favoring the 7 Repeat Allele of VNTR in the DRD4 Gene. PLoS ONE 6(8): e24410. https://doi.org/10.1371/journal.pone.0024410

References

1. Van Tol HH, Wu CM, Guan HC, Ohara K, Bunzow JR, et al. (1992) Multiple dopamine D4 receptor variants in the human population. Nature 358: 149–152.HH Van TolCM WuHC GuanK. OharaJR Bunzow1992Multiple dopamine D4 receptor variants in the human population.Nature358149152

2. Chang FM, Kidd JR, Livak KJ, Pakstis AJ, Kidd KK (1996) The world-wide distribution of allele frequencies at the human dopamine D4 receptor locus. Hum Genet 98: 91–101.FM ChangJR KiddKJ LivakAJ PakstisKK Kidd1996The world-wide distribution of allele frequencies at the human dopamine D4 receptor locus.Hum Genet9891101

3. Asghari V, Sanyal S, Buchwaldt S, Paterson A, Jovanovic V, et al. (1995) Modulation of intracellular cyclic AMP levels by different human dopamine D4 receptor variants. J Neurochem 65: 1157–1165.V. AsghariS. SanyalS. BuchwaldtA. PatersonV. Jovanovic1995Modulation of intracellular cyclic AMP levels by different human dopamine D4 receptor variants.J Neurochem6511571165

4. Schoots O, Van Tol HH (2003) The human dopamine D4 receptor repeat sequences modulate expression. Pharmacogenomics J 3: 343–348.O. SchootsHH Van Tol2003The human dopamine D4 receptor repeat sequences modulate expression.Pharmacogenomics J3343348

5. Ebstein RP, Novick O, Umansky R, Priel B, Osher Y, et al. (1996) Dopamine D4 receptor (D4DR) exon III polymorphism associated with the human personality trait of Novelty Seeking. Nat Genet 12: 78–80.RP EbsteinO. NovickR. UmanskyB. PrielY. Osher1996Dopamine D4 receptor (D4DR) exon III polymorphism associated with the human personality trait of Novelty Seeking.Nat Genet127880

6. Benjamin J, Li L, Patterson C, Greenberg BD, Murphy DL, et al. (1996) Population and familial association between the D4 dopamine receptor gene and measures of Novelty Seeking. Nat Genet 12: 81–84.J. BenjaminL. LiC. PattersonBD GreenbergDL Murphy1996Population and familial association between the D4 dopamine receptor gene and measures of Novelty Seeking.Nat Genet128184

7. LaHoste GJ, Swanson JM, Wigal SB, Glabe C, Wigal T, et al. (1996) Dopamine D4 receptor gene polymorphism is associated with attention deficit hyperactivity disorder. Mol Psychiatry 1: 121–124.GJ LaHosteJM SwansonSB WigalC. GlabeT. Wigal1996Dopamine D4 receptor gene polymorphism is associated with attention deficit hyperactivity disorder.Mol Psychiatry1121124

8. Faraone SV, Doyle AE, Mick E, Biederman J (2001) Meta-analysis of the association between the 7-repeat allele of the dopamine D(4) receptor gene and attention deficit hyperactivity disorder. Am J Psychiatry 158: 1052–1057.SV FaraoneAE DoyleE. MickJ. Biederman2001Meta-analysis of the association between the 7-repeat allele of the dopamine D(4) receptor gene and attention deficit hyperactivity disorder.Am J Psychiatry15810521057

9. Malhotra AK, Virkkunen M, Rooney W, Eggert M, Linnoila M, et al. (1996) The association between the dopamine D4 receptor (D4DR) 16 amino acid repeat polymorphism and novelty seeking. Mol Psychiatry 1: 388–391.AK MalhotraM. VirkkunenW. RooneyM. EggertM. Linnoila1996The association between the dopamine D4 receptor (D4DR) 16 amino acid repeat polymorphism and novelty seeking.Mol Psychiatry1388391

10. Gelernter J, Kranzler H, Coccaro E, Siever L, New A, et al. (1997) D4 dopamine-receptor (DRD4) alleles and novelty seeking in substance-dependent, personality-disorder, and control subjects. Am J Hum Genet 61: 1144–1152.J. GelernterH. KranzlerE. CoccaroL. SieverA. New1997D4 dopamine-receptor (DRD4) alleles and novelty seeking in substance-dependent, personality-disorder, and control subjects.Am J Hum Genet6111441152

11. Swanson J, Oosterlaan J, Murias M, Schuck S, Flodman P, et al. (2000) Attention deficit/hyperactivity disorder children with a 7-repeat allele of the dopamine receptor D4 gene have extreme behavior but normal performance on critical neuropsychological tests of attention. Proc Natl Acad Sci U S A 97: 4754–4759.J. SwansonJ. OosterlaanM. MuriasS. SchuckP. Flodman2000Attention deficit/hyperactivity disorder children with a 7-repeat allele of the dopamine receptor D4 gene have extreme behavior but normal performance on critical neuropsychological tests of attention.Proc Natl Acad Sci U S A9747544759

12. Kotler M, Manor I, Sever Y, Eisenberg J, Cohen H, et al. (2000) Failure to replicate an excess of the long dopamine D4 exon III repeat polymorphism in ADHD in a family-based study. Am J Med Genet 96: 278–281.M. KotlerI. ManorY. SeverJ. EisenbergH. Cohen2000Failure to replicate an excess of the long dopamine D4 exon III repeat polymorphism in ADHD in a family-based study.Am J Med Genet96278281

13. Castellanos FX, Lau E, Tayebi N, Lee P, Long RE, et al. (1998) Lack of an association between a dopamine-4 receptor polymorphism and attention-deficit/hyperactivity disorder: genetic and brain morphometric analyses. Mol Psychiatry 3: 431–434.FX CastellanosE. LauN. TayebiP. LeeRE Long1998Lack of an association between a dopamine-4 receptor polymorphism and attention-deficit/hyperactivity disorder: genetic and brain morphometric analyses.Mol Psychiatry3431434

14. Ding YC, Chi HC, Grady DL, Morishima A, Kidd JR, et al. (2002) Evidence of positive selection acting at the human dopamine receptor D4 gene locus. Proc Natl Acad Sci U S A 99: 309–314.YC DingHC ChiDL GradyA. MorishimaJR Kidd2002Evidence of positive selection acting at the human dopamine receptor D4 gene locus.Proc Natl Acad Sci U S A99309314

15. Hattori E, Nakajima M, Yamada K, Iwayama Y, Toyota T, et al. (2009) Variable number of tandem repeat polymorphisms of DRD4: re-evaluation of selection hypothesis and analysis of association with schizophrenia. Eur J Hum Genet 17: 793–801.E. HattoriM. NakajimaK. YamadaY. IwayamaT. Toyota2009Variable number of tandem repeat polymorphisms of DRD4: re-evaluation of selection hypothesis and analysis of association with schizophrenia.Eur J Hum Genet17793801

16. Wang E, Ding YC, Flodman P, Kidd JR, Kidd KK, et al. (2004) The genetic architecture of selection at the human dopamine receptor D4 (DRD4) gene locus. Am J Hum Genet 74: 931–944.E. WangYC DingP. FlodmanJR KiddKK Kidd2004The genetic architecture of selection at the human dopamine receptor D4 (DRD4) gene locus.Am J Hum Genet74931944

17. Ohashi J, Naka I, Patarapotikul J, Hananantachai H, Brittenham G, et al. (2004) Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection. Am J Hum Genet 74: 1198–1208.J. OhashiI. NakaJ. PatarapotikulH. HananantachaiG. Brittenham2004Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection.Am J Hum Genet7411981208

18. Sabeti PC, Reich DE, Higgins JM, Levine HZ, Richter DJ, et al. (2002) Detecting recent positive selection in the human genome from haplotype structure. Nature 419: 832–837.PC SabetiDE ReichJM HigginsHZ LevineDJ Richter2002Detecting recent positive selection in the human genome from haplotype structure.Nature419832837

19. Tishkoff SA, Varkonyi R, Cahinhinan N, Abbes S, Argyropoulos G, et al. (2001) Haplotype diversity and linkage disequilibrium at human G6PD: recent origin of alleles that confer malarial resistance. Science 293: 455–462.SA TishkoffR. VarkonyiN. CahinhinanS. AbbesG. Argyropoulos2001Haplotype diversity and linkage disequilibrium at human G6PD: recent origin of alleles that confer malarial resistance.Science293455462

20. The International HapMap consortium (2003) The International HapMap Project. Nature 426: 789–796.The International HapMap consortium2003The International HapMap Project.Nature426789796

21. The International HapMap consortium (2005) A haplotype map of the human genome. Nature 437: 1299–1320.The International HapMap consortium2005A haplotype map of the human genome.Nature43712991320

22. Voight BF, Kudaravalli S, Wen X, Pritchard JK (2006) A map of recent positive selection in the human genome. PLoS Biol 4: e72.BF VoightS. KudaravalliX. WenJK Pritchard2006A map of recent positive selection in the human genome.PLoS Biol4e72

23. Watterson GA (1978) The homozygosity test of neutrality. Genetics 88: 405–417.GA Watterson1978The homozygosity test of neutrality.Genetics88405417

24. Ohashi J, Naka I, Tsuchiya N (2011) The impact of natural selection on an ABCC11 SNP determining earwax type. Mol Biol Evol 28: 849–857.J. OhashiI. NakaN. Tsuchiya2011The impact of natural selection on an ABCC11 SNP determining earwax type.Mol Biol Evol28849857

25. Sabeti PC, Walsh E, Schaffner SF, Varilly P, Fry B, et al. (2005) The case for selection at CCR5-Delta32. PLoS Biol 3: e378.PC SabetiE. WalshSF SchaffnerP. VarillyB. Fry2005The case for selection at CCR5-Delta32.PLoS Biol3e378

26. Spencer CC, Coop G (2004) SelSim: a program to simulate population genetic data with natural selection and recombination. Bioinformatics 20: 3673–3675.CC SpencerG. Coop2004SelSim: a program to simulate population genetic data with natural selection and recombination.Bioinformatics2036733675

27. Saunders MA, Slatkin M, Garner C, Hammer MF, Nachman MW (2005) The extent of linkage disequilibrium caused by selection on G6PD in humans. Genetics 171: 1219–1229.MA SaundersM. SlatkinC. GarnerMF HammerMW Nachman2005The extent of linkage disequilibrium caused by selection on G6PD in humans.Genetics17112191229

28. Livak KJ, Rogers J, Lichter JB (1995) Variability of dopamine D4 receptor (DRD4) gene sequence within and among nonhuman primate species. Proc Natl Acad Sci U S A 92: 427–431.KJ LivakJ. RogersJB Lichter1995Variability of dopamine D4 receptor (DRD4) gene sequence within and among nonhuman primate species.Proc Natl Acad Sci U S A92427431

29. Qian Q, Wang Y, Zhou R, Yang L, Faraone SV (2004) Family-based and case-control association studies of DRD4 and DAT1 polymorphisms in Chinese attention deficit hyperactivity disorder patients suggest long repeats contribute to genetic risk for the disorder. Am J Med Genet B Neuropsychiatr Genet 128B: 84–89.Q. QianY. WangR. ZhouL. YangSV Faraone2004Family-based and case-control association studies of DRD4 and DAT1 polymorphisms in Chinese attention deficit hyperactivity disorder patients suggest long repeats contribute to genetic risk for the disorder.Am J Med Genet B Neuropsychiatr Genet128B8489

30. Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform Online 1: 47–50.L. ExcoffierG. LavalS. Schneider2005Arlequin (version 3.0): an integrated software package for population genetics data analysis.Evol Bioinform Online14750

31. Ewens WJ (1972) The sampling theory of selectively neutral alleles. Theor Popul Biol 3: 87–112.WJ Ewens1972The sampling theory of selectively neutral alleles.Theor Popul Biol387112

32. Wright S (1951) The genetical structure of populations. Ann Eugen 15: 323–354.S. Wright1951The genetical structure of populations.Ann Eugen15323354

33. Barrett JC, Fry B, Maller J, Daly MJ (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21: 263–265.JC BarrettB. FryJ. MallerMJ Daly2005Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics21263265

34. Scheet P, Stephens M (2006) A fast and flexible statistical model for large-scale population genotype data: applications to inferring missing genotypes and haplotypic phase. Am J Hum Genet 78: 629–644.P. ScheetM. Stephens2006A fast and flexible statistical model for large-scale population genotype data: applications to inferring missing genotypes and haplotypic phase.Am J Hum Genet78629644