Recent scans of genome-wide polymorphism data have generated long lists of genomic regions that are believed to have been targeted by local positive selection, but few of these regions have been shown to harbor functional variants or to be linked to a putatively adaptive phenotype. Detailed investigation of these candidate genomic regions is required for a comprehensive picture of local human adaptation. The present study provides an analysis of genetic variation and function at the EDAR
gene, a candidate for positive selection in East Asians identified from recent genome scans 
We provide two lines of evidence supporting the hypothesis that the derived 370A allele in EDAR
is functional and experienced positive selection in East Asians. First, worldwide population differentiation for 370A as measured by Fst is highly unusual. demonstrates that 370A has a highly unusual worldwide frequency distribution, supporting a scenario in which the 370A allele was driven to high frequency in East Asians and Native Americans by positive selection (). Two Central/South Asia populations, the Uygur and the Hazara, have intermediate frequencies of 370A (0.44 and 0.5, respectively), in agreement with their close genetic relationship to East Asians 
. The 370A allele is also found at low frequency in Melanesia (0.12), and was likely introduced there via the recent Austronesian expansion 
. Otherwise 370A is absent in Africans and Papuans and is observed at very low frequency in most of Central/South Asia, Europe and the Middle East ().
The second line of evidence suggesting an adaptive functional role for 370A stems from its location in the amino acid sequence of EDAR. V370A is located in the death domain, a protein interaction module, of EDAR. The death domain of EDAR is highly conserved () and interacts with the death domain of EDARADD, an intracellular ligand to EDAR 
. This interaction initiates an intracellular signalling cascade that results in the activation of the transcription factor NF-κB 
. Therefore, V370A may alter binding affinity with the death domain of EDARADD and thereby influence the activation of NF-κB. Moreover, seven nonsynonymous substitutions in the death domain of EDAR cause hypohidrotic ectodermal dysplasia (HED) in humans 
, a disease characterized by sparse and thin hair, missing teeth and the absence of sweat glands (OMIM
604095; see ). In particular, the R375H substitution, only 5 amino acids upstream from V370A, results in a loss of affinity for EDARADD and reduced NF-κB activation 
. We therefore tested the function of V370A in vitro and found that the 370A allele does differ from the ancestral 370V allele in that it results in enhanced NF-κB activation ().
Our results contradict a recent report from Fujimoto et al. 
in which the 370A allele was shown to reduce NF-κB activation in vitro 
. We are confident that our results are correct for two reasons. First, in addition to observing enhanced NF-κB activation for the derived 370A allele on a normal genetic background, we measured NF-κB activation of the 370V and 370A alleles on the background of a disease mutation (375H) that was previously demonstrated to result in significantly reduced NF-κB activation 
. In agreement with 
, we observed significantly reduced NF-κB activation in both clones carrying the 375H disease mutation. Moreover, NF-κB activation was significantly higher in the derived 370A+375H construct than in the ancestral 370V+375H construct (). Thus, we observed enhanced NF-κB activation for the derived 370A allele in two independent constructs. Second, East Asians have thicker hair than Europeans and Africans 
and an increase in NF-κB activation is arguably more likely to lead to the thicker East Asian hair phenotype. This is because a decrease in NF-κB activation, as observed for carriers of the 375H allele that causes hypohidrotic ectodermal dysplasia, is associated with thin hair. We suspect that the large doses of plasmid DNA (300 ng) and long post-transfection incubation period (48 h) could have induced cell death in the experiments of Fujimoto et al. 
. Although EDAR-induced cell death is a matter of controversy 
, several features of cell death (detachment, rounding and membrane permeation) are observed 36 hours after transfection with a high dosage of plasmid (500 ng) 
. Control cells transfected with the same amount of an empty vector do not display features of cell death, suggesting that simply overdosing the cells with plasmid is not responsible for cell death 
. Thus, we speculate that the reduced NF-κB activation from 370A observed by Fujimoto et al. 
could be the result of induced cell death and that 370A in fact enhances NF-κB activation.
Our estimate of the time since fixation of 370A in a sample of 45 Chinese chromosomes is 10,740 years (). This estimate involves several assumptions (see Materials and Methods
) and should be interpreted with caution. Nevertheless the result suggests that 370A was likely at high frequency before the colonization of the Americas 10,500–14,000 years ago 
. Thus, the high frequency of 370A in Native Americans (see ) is most likely due to positive selection prior to migrations from Asia to America.
In summary, we have demonstrated that the worldwide frequency distribution of 370A is highly unusual and that 370A was likely rising in frequency by positive selection in East Asia prior to 10,000 years ago. In addition, we have shown that the 370A allele results in enhanced NF-κB activation in vitro. What was the source of the selection pressure on 370A and what effect may 370A have on the phenotype? Since EDAR is involved in ectodermal development, 370A might be expected to affect teeth, hair, skin, nails and/or sweat glands. Fujimoto et al. 
recently noted an association between 370A and hair thickness. Replication of this result is desirable since correction for population structure was inadequate: only a single SNP was used to correct for population structure. Nevertheless, the results of Fujimoto et al. 
are suggestive, especially since East Asians have thicker hair than Europeans and Africans 
. These observations lead us to question why thicker hair may have been advantageous in ancestral East Asian environments. Of course, thicker hair may not have been adaptive at all and may simply be the result of phenotypic hitchhiking: selection on 370A may have targeted a different phenotype (e.g. tooth morphology 
) and hair thickness may have resulted as a by-product of this selection. Sexual selection also remains a possibility.
Regardless of the nature of the selective force, our results provide compelling evidence that positive selection has acted on the 370A allele in EDAR. In addition, our finding that 370A results in increased NF-κB activation suggests further lines for investigation: in particular, how does this increased NF-κB activation influence the expression of the target genes regulated by NF-κB? Such future studies will lead to a more complete understanding of the phenotypic effect of 370A, and will permit more explicit tests of hypotheses concerning the possible selection pressure(s) responsible for its rapid increase in frequency in East Asia.