Cel Meibomian Glands selected in this scheme can be seen as a random sampling for the 370A gland preparations were made from the left eyelids were fixed flat on Whatman paper in 4% Paraf Detailed phenotype collection and calling procedure as well as statistical er), and photographed on a Leica MZFLIll ster methods are provided in Association Study of 370A in a Han Chinese Popula quipped with a Nikon DXM1200F camera Total glandular area w tion. We performed all human subjects work in accordance with approved om images using ImageJ (v 1.46, (Schneider et al., 2012)-3 protocols by Fudan and Harvard Universities. 370V/370A (n= 25), and 370A (n= 20)animals were analyzed SUPPLEMENTAL INFORMATION The 4th and gh inguinal mammary glands and associated fat pads were supplement si t ental Proce issected from 6-week-old, virgin female mice. Whole mount preparations figuresandsixtablesandcanbefoundwiththisarticleonlineathttp:/dx.doi mammary glands and staining of the ductal tree were performed as org/10.1016cel201301016 /ndex html Mammary glands were fixed flat in Carnoy's fix then stained with carmine alum(Sigma Aldrich). Stained glands were dehydrated into Xylenes (Thermo ACKNOWLEDGMENTS Fisher), mounted flat, and photographed on a Leica MZFLIll s quipped with a Nikon DXM1200F camera. Several high-resolution images of This project was funded by a grant from the Harvard University Science and each gland were merged for analysis in Photoshop (Adobe Systems ). Engineering Committee Seed Fund for Interdisciplinary Science to CJT, ce and Engi- were measured from the main lactiferous duct to the dorsolateral edge of the neenng and an NIH Innovator Award 1DP20D006514-01 to PCS: a BIRT gland Gland length was measured between the distal-most ductal termini at Award AR055256-04S1 from NIAMS to BAM; an NIH grant R37 HD032443 her end of the gland. Total branch number was assessed by counting all CJT; and funding from the american School of Prehistoric Research to uctal termini per gland using the Image Cell Counter plug- DEL Human association study work was additionally supported by NSFC was calculated by dividing total branch number by gland length. Left and 30890034: MOST 2011BAI09B00: MOH 201002007 to LJ. YI was funded by right glands of each animal were averaged together. 370v (n 10), 370v/ an AXA Research Fund postdoctoral fellowship and PG by the LeCHE Marie 70A (n= 19), and 370A(n= 11)animals were assessed R37 054364 grant. We acknowledge the UCL Legion High Performance Mouse Eccrine Glands omputing Facility and support services and thank O. Bar Yosef, C. Zhao E. Rohling, S Schaffner, L. Gaffney, C. Edwards, J. Vitti, S Tabrizi and A Tariel skin from both hindfeet and ating in Dispase ll(Roche)as described for feedback on the manuscript and A Carpenter, C walby, and M Moro previously(Okada et al, 1983).Next, the epidermis was peeled away from for data analysis help. The authors declare no conflict of interest the underlying demis. Eccrine gland ducts remained associated with the 21,2012 ns Revised: November 22.2012 also stained with 0.5% Oil Red O(Sigma), which stains sebaceous Accepted: January 4, 2013 glands. Footpads 1 and 2 were not analyzed because their high eccrine gland Published: February 14, 2013 density prevents accurate scoring. The number of eccrine glands per footpad indfeet. 370V(n= 17), 370V/370A(n= 18), and REFERENCES 70A(n= 16)animals were assessed to evaluate the effect of 370A on eccrine gland number and 370V/379E(n= 12), 370V/379K (n= 11), and 370A/379K(n= Akey, JM.(2009). humans: where do we go from here? Genome Res. 19, 711-722. Details of all statistical tests of mouse data are reported in Generation and Philippe, P, Raphael, D, Stini, W.A., and Esterik, P v (1983 ). The Reproduc Beaumont, M.A, Zhang W, and Balding, D.J.(2002). Approximate Bayesian Association Study computation in population genetics. Genetics 162, 2025-2035 Population le studied a Han chinese Bertorelle, G, Benazzo, A, and Mona, S(2010. ABC as a flexible framework te demography over space and time some cons, many pros. MoL. from five closely located villages in Taizhou(ages 35-65) and local students of C Taizhou Professional Technology College(ages 18-21). All participants spent Bramble, D M, and Lieberman, D.E. (2004). Endurance running and the evolu- the majority or entirety of their lives in Taizhou and are expected to be tion of Homo. Nature 432, 345-352 Hardouin, E, A., DNA Extraction, Genotyping, and Sample and Myles, S.(2008). Positive selection in East As EDAR allele DNA extraction and at Fudan ur ity. Upon that enhances NF-kappaB activation. PLOS ONE 3, e2209. enrollment in the cohort, each participant's blood samples collected el, V, Piouffre, L, and stored in the cohort database. DNA was isolated using standard phenol Bodmer. J. Bodmer W.F. Bonne-Tamir. B. Cambon-Thomsen. A. et al hloroform extraction. The EDAR SNP,rs3827760, was genotyped using the (2002 ). A human genome diversity cell line panel. Science 296, 261-262 NaPshot Multiplex System which included seven other SNPs that showed signatures of positive selection in East Asia. Genotype calling was performe Carrier, D R, Kapoor, A K, Kimura, T, Nickels, MK, Satwanti, Scott, E.C. y GeneMapper v2.0. We compiled a priority list of potential study participan So, J K, and Trinkaus, E(1984). The Energetic Paradox of Human Running ased on genotype results. 370V allele cariers had top priority, followed by the and Hominid Evolution. Curr. Anthropol. 25, 483-495. allele carriers of the other seven SNPs. From the 2, 572 samples geno. Carroll, SB(2008). EVO-devo and an expanding typed, we contacted the top 1, 000 individuals, and enrolled 623 in this study n Cell 13 (427 from the villages and 196 from the college). Among the other SNPs Chang, S.H., Jobling, S, Brennan, K, and Headon, Enhanced genotyped, none are on the same chromosome as 370A, and none were asso- Edar signalling has pleiotropic effects on craniofacial ciated with 370A. Therefore. we concluded that the 437 370A individuals PLos oNE 4. 07591 700cel152,691-702, February14,2013e2013EsMeibomian Glands Meibomian gland preparations were made from the left eyelids of 6-week-old mice. Eyelids were fixed flat on Whatman paper in 4% Paraformaldehyde (Thermo Fisher), and photographed on a Leica MZFLIII stereomicroscope equipped with a Nikon DXM1200F camera. Total glandular area was measured from images using ImageJ (v.1.46, (Schneider et al., 2012)). 370V (n = 13), 370V/370A (n = 25), and 370A (n = 20) animals were analyzed. Mammary Glands The 4th and 9th inguinal mammary glands and associated fat pads were dissected from 6-week-old, virgin female mice. Whole mount preparations of mammary glands and staining of the ductal tree were performed as described (http://mammary.nih.gov/tools/histological/Histology/index.html#a1). Mammary glands were fixed flat in Carnoy’s fix then stained with carmine alum (Sigma Aldrich). Stained glands were dehydrated into Xylenes (Thermo Fisher), mounted flat, and photographed on a Leica MZFLIII stereomicroscope equipped with a Nikon DXM1200F camera. Several high-resolution images of each gland were merged for analysis in Photoshop (Adobe Systems). Image analysis was performed with ImageJ. Fat pad area and glandular area were measured from the main lactiferous duct to the dorsolateral edge of the gland. Gland length was measured between the distal-most ductal termini at either end of the gland. Total branch number was assessed by counting all ductal termini per gland using the ImageJ Cell Counter plug-in. Branch density was calculated by dividing total branch number by gland length. Left and right glands of each animal were averaged together. 370V (n = 10), 370V/ 370A (n = 19), and 370A (n = 11) animals were assessed. Mouse Eccrine Glands Epidermal whole-mount preparations were prepared by dissecting the volar skin from both hindfeet and incubating in Dispase II (Roche) as described previously (Okada et al., 1983). Next, the epidermis was peeled away from the underlying dermis. Eccrine gland ducts remained associated with the epidermis and were stained with a 0.1% solution of Nile Blue A (Sigma Aldrich) and observed on a Leica MZFLIII stereomicroscope. Epidermal preparations were also stained with 0.5% Oil Red O (Sigma), which stains sebaceous glands. Footpads 1 and 2 were not analyzed because their high eccrine gland density prevents accurate scoring. The number of eccrine glands per footpad was averaged across both hindfeet. 370V (n = 17), 370V/370A (n = 18), and 370A (n = 16) animals were assessed to evaluate the effect of 370A on eccrine gland number and 370V/379E (n = 12), 370V/379K (n = 11), and 370A/379K (n = 13) animals were analyzed to evaluate the effect of 370A on the 379K mutation in separate crosses. Glands were analyzed from mice aged 3 to 6 weeks. Details of all statistical tests of mouse data are reported in Generation and Statistical Analysis of the 370A Knockin Mouse and Table S5. Association Study Population We studied a Han Chinese population from an established Taizhou longitudinal cohort in Jiangsu Province, China (Wang et al., 2009), that recruited individuals from five closely located villages in Taizhou (ages 35–65) and local students of Taizhou Professional Technology College (ages 18–21). All participants spent the majority or entirety of their lives in Taizhou and are expected to be homogeneous. DNA Extraction, Genotyping, and Sample Selection DNA extraction and genotyping were performed at Fudan University. Upon enrollment in the cohort, each participant’s blood samples was collected and stored in the cohort database. DNA was isolated using standard phenol/ chloroform extraction. The EDAR SNP, rs3827760, was genotyped using the SNaPshot Multiplex System which included seven other SNPs that showed signatures of positive selection in East Asia. Genotype calling was performed by GeneMapper v2.0. We compiled a priority list of potential study participants based on genotype results. 370V allele carriers had top priority, followed by the rare allele carriers of the other seven SNPs. From the 2,572 samples geno￾typed, we contacted the top 1,000 individuals, and enrolled 623 in this study (427 from the villages and 196 from the college). Among the other SNPs genotyped, none are on the same chromosome as 370A, and none were asso￾ciated with 370A. Therefore, we concluded that the 437 370A individuals selected in this scheme can be seen as a random sampling for the 370A association study. Detailed phenotype collection and calling procedure as well as statistical methods are provided in Association Study of 370A in a Han Chinese Popula￾tion. We performed all human subjects work in accordance with approved protocols by Fudan and Harvard Universities. SUPPLEMENTAL INFORMATION Supplemental Information includes Extended Experimental Procedures, seven figures, and six tables and can be found with this article online at http://dx.doi. org/10.1016/j.cell.2013.01.016. ACKNOWLEDGMENTS This project was funded by a grant from the Harvard University Science and Engineering Committee Seed Fund for Interdisciplinary Science to CJT, PCS, BAM and DEL; a Packard Foundation Fellowship in Science and Engi￾neering and an NIH Innovator Award 1DP2OD006514-01 to PCS; a BIRT Award AR055256-04S1 from NIAMS to BAM; an NIH grant R37 HD032443 to CJT; and funding from the American School of Prehistoric Research to DEL. Human association study work was additionally supported by NSFC 30890034; MOST 2011BAI09B00; MOH 201002007 to LJ. YI was funded by an AXA Research Fund postdoctoral fellowship and PG by the LeCHE Marie Curie FP7 framework. Work in APM’s laboratory was supported by an NIH R37 054364 grant. We acknowledge the UCL Legion High Performance Computing Facility and support services and thank O. Bar Yosef, C. Zhao, E. Rohling, S. Schaffner, L.Gaffney, C. Edwards, J. Vitti, S.Tabrizi and A.Tariela for feedback on the manuscript and A.Carpenter, C. Wa¨ hlby, and M. Morgan for data analysis help. The authors declare no conflict of interest. Received: September 21, 2012 Revised: November 22, 2012 Accepted: January 4, 2013 Published: February 14, 2013 REFERENCES Akey, J.M. (2009). Constructing genomic maps of positive selection in humans: where do we go from here? Genome Res. 19, 711–722. Anderson, P., Frisch, R.E., Graham, C.E., Manderson, L., Orubuloye, I.O., Philippe, P., Raphael, D., Stini, W.A., and Esterik, P.V. (1983). The Reproduc￾tive Role of the Human Breast. Curr. Anthropol. 24, 25–45. Beaumont, M.A., Zhang, W., and Balding, D.J. (2002). Approximate Bayesian computation in population genetics. Genetics 162, 2025–2035. Bertorelle, G., Benazzo, A., and Mona, S. (2010). ABC as a flexible framework to estimate demography over space and time: some cons, many pros. Mol. Ecol. 19, 2609–2625. Bramble, D.M., and Lieberman, D.E. (2004). Endurance running and the evolu￾tion of Homo. Nature 432, 345–352. Bryk, J., Hardouin, E., Pugach, I., Hughes, D., Strotmann, R., Stoneking, M., and Myles, S. (2008). Positive selection in East Asians for an EDAR allele that enhances NF-kappaB activation. PLoS ONE 3, e2209. Cann, H.M., de Toma, C., Cazes, L., Legrand, M.-F., Morel, V., Piouffre, L., Bodmer, J., Bodmer, W.F., Bonne-Tamir, B., Cambon-Thomsen, A., et al. (2002). A human genome diversity cell line panel. Science 296, 261–262. Carrier, D.R., Kapoor, A.K., Kimura, T., Nickels, M.K., Satwanti, Scott, E.C., So, J.K., and Trinkaus, E. (1984). The Energetic Paradox of Human Running and Hominid Evolution. Curr. Anthropol. 25, 483–495. Carroll, S.B. (2008). Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 134, 25–36. Chang, S.H., Jobling, S., Brennan, K., and Headon, D.J. (2009). Enhanced Edar signalling has pleiotropic effects on craniofacial and cutaneous glands. PLoS ONE 4, e7591. 700 Cell 152, 691–702, February 14, 2013 ª2013 Elsevier Inc