Cell zygotes(MANOVA post hoc tests, p=0.002, Figure 6H). In agreement with a gain-of-function model, 370A/379K heterozy (p < 0.05 for all footpads, Figure 6H and Table S5) 370A Is Associated with More Eccrine Glands and other Pleiotropic Effects in Humans 4 cell 3 cell 2 cell The change in eccrine gland number we observed in the 370A mouse has important implications for the distribution of variation in this trait in human populations. However, association studies of sweat gland density with nonpathological variation at the EDAR locus have not been reported in humans. To examine whether 370A is associated with altered eccrine gland number in humans, we carried out an association study 82 in individuals of han descent from an established cohort in taiz- 370V hou, China Wang et al., 2009). To sample a sufficient number of 370v/370A the rare 370V alleles, we first genotyped the 370A SNP and found 2, 226 370A homozygotes, 340 370V/370A heterozygotes, and 6 370V homozygotes. We then contacted all individuals with at least one copy of the 370V allele and enrolled 187 of them(184 370V/370A and 3 3701, along with 436 370A individuals and collected phenotypes related to ectodermal appendages ( Table S6 and Association Study of 370A in a Han Chinese Population) Because only three individuals were homozygous for the 370V Number of cells for 370A in statistical analysis of the collected data Consistent with previous reports(Kimura et al., 2009: Park et aL., 2012), 370A was associated with single and doub shoveling of the upper incisors (Wald test, p= 0.0077 and p=0.0004, respectively; Table S6). Additionally, 370A was significantly associated with the presence of a protostylid cusp and the absence of lower third molars (Wald test, p=0.0079 ■370V and p=0.0123, respectively; Association Study of 370A in a 370V/370A Han Chinese Population and Table S6) We tested for an association between 370A and eccrine sweat gland number using the starch-iodine method to measure the number of activated glands in digit pads of the thumb and inde finger(Juniper et aL., 1964: Randall, 1946 ). In agreement with our mouse findings, 370A homozygous individuals had significantly more active eccrine glands than 370V/370A individuals(two- ailed t test, p=0.011, Figure 7). Testing all three genotypes Number of cells sing linear-regression in an additive model revealed a strong Figure 3. 370A Allele Increases Hair Size in the Mouse Coat association between 370A and eccrine gland density ( Wald (A) Representative images of hairs show medulla cell number serves test, p=0.0047; Table S6). This association remained significant a proxy for hair shaft thicknes when we controlled for age, sex, and potential population (B and C)370A mice have a larger proportion of thicker hairs than mice substructure(Association Study of 370A in a Han Chinese pressing the ancestral allele the average frequency (+SEM)of awl( B)and Population and Table S6). DISCUSSION post hoc tests: p<0.05(),p<0.01("). See also Table $5. study integrated population genetic analyses, a humanized the eccrine gland phenotype of mice heterozygous for the mouse model, and human association study to characterize downless(dl,E379K@Edarloss-of-functionmutation(Headonanaturalhumangenevariant.Combiningtheseapproache and Overbeek, 1999). 379K is classically considered a recessive allowed us to determine the direct biological effects of 370A mutation, and animals heterozygous for the 379K allele are and cast new light on their evolutionary uences Extend- described as wild-type(Headon and Overbeek, 1999). However, ing this strategy to other candidate adaptive alleles stands to our quantitative method for scoring eccrine glands revealed advance our understanding of the effects of recent selection a subtle decrease in eccrine gland number in 379E/379K hetero- on the diversification of modern humans. Cell 152, 691-702, February 14, 2013(2013 Elsevier Inc. 695the eccrine gland phenotype of mice heterozygous for the downless (dlj , E379K) Edar loss-of-function mutation (Headon and Overbeek, 1999). 379K is classically considered a recessive mutation, and animals heterozygous for the 379K allele are described as wild-type (Headon and Overbeek, 1999). However, our quantitative method for scoring eccrine glands revealed a subtle decrease in eccrine gland number in 379E/379K hetero￾zygotes (MANOVA post hoc tests, p = 0.002, Figure 6H). In agreement with a gain-of-function model, 370A/379K heterozy￾gous animals had more eccrine glands than 370V/379K animals (p < 0.05 for all footpads, Figure 6H and Table S5). 370A Is Associated with More Eccrine Glands and Other Pleiotropic Effects in Humans The change in eccrine gland number we observed in the 370A mouse has important implications for the distribution of variation in this trait in human populations. However, association studies of sweat gland density with nonpathological variation at the EDAR locus have not been reported in humans. To examine whether 370A is associated with altered eccrine gland number in humans, we carried out an association study in individuals of Han descent from an established cohort in Taiz￾hou, China (Wang et al., 2009). To sample a sufficient number of the rare 370V alleles, we first genotyped the 370A SNP and found 2,226 370A homozygotes, 340 370V/370A heterozygotes, and 6 370V homozygotes. We then contacted all individuals with at least one copy of the 370V allele and enrolled 187 of them (184 370V/370A and 3 370V), along with 436 370A individuals and collected phenotypes related to ectodermal appendages (Table S6 and Association Study of 370A in a Han Chinese Population). Because only three individuals were homozygous for the 370V allele, we focused on individuals homozygous and heterozygous for 370A in statistical analysis of the collected data. Consistent with previous reports (Kimura et al., 2009; Park et al., 2012), 370A was associated with single and double shoveling of the upper incisors (Wald test, p = 0.0077 and p = 0.0004, respectively; Table S6). Additionally, 370A was significantly associated with the presence of a protostylid cusp and the absence of lower third molars (Wald test, p = 0.0079 and p = 0.0123, respectively; Association Study of 370A in a Han Chinese Population and Table S6). We tested for an association between 370A and eccrine sweat gland number using the starch-iodine method to measure the number of activated glands in digit pads of the thumb and index finger (Juniper et al., 1964; Randall, 1946). In agreement with our mouse findings, 370A homozygous individuals had significantly more active eccrine glands than 370V/370A individuals (two￾tailed t test, p = 0.011, Figure 7). Testing all three genotypes using linear-regression in an additive model revealed a strong association between 370A and eccrine gland density (Wald test, p = 0.0047; Table S6). This association remained significant when we controlled for age, sex, and potential population substructure (Association Study of 370A in a Han Chinese Population and Table S6). DISCUSSION This study integrated population genetic analyses, a humanized mouse model, and human association study to characterize a natural human gene variant. Combining these approaches allowed us to determine the direct biological effects of 370A and cast new light on their evolutionary consequences. Extend￾ing this strategy to other candidate adaptive alleles stands to advance our understanding of the effects of recent selection on the diversification of modern humans. Figure 3. 370A Allele Increases Hair Size in the Mouse Coat (A) Representative images of mouse hairs show medulla cell number serves as a proxy for hair shaft thickness. (B and C) 370A mice have a larger proportion of thicker hairs than mice expressing the ancestral allele. The average frequency (±SEM) of awl (B) and auchene (C) hairs of each size is shown. Significance levels of differences between 370V and 370A animals by ANOVA post hoc tests: p < 0.05 (*), p < 0.01 (**). See also Table S5. Cell 152, 691–702, February 14, 2013 ª2013 Elsevier Inc. 695