ARTICLE Received 18 Aug 2014 | Accepted 27 Jan 2015 | Published 16 Mar 2015 DOI: 10.1038/ncomms7414 Genome-wide association study identifies a new susceptibility locus for cleft lip with or without a cleft palate Yimin Sun1,2,3,4,*, Yongqing Huang5,6,*, Aihua Yin7,8,9,*, Yongchu Pan10,11,12,*, Yirui Wang1,3,4,*, Cheng Wang12,13,*, Yong Du5,6, Meilin Wang14, Feifei Lan7,8,9, Zhibin Hu12,13, Guoqing Wang3,4, Min Jiang5,6, Junqing Ma10,11, Xiaozhuang Zhang15, Hongxia Ma12,13, Jian Ma5,6, Weibing Zhang10,11, Qun Huang16, Zhongwei Zhou5,6, Lan Ma10,11, Yadi Li5,6, Hongbing Jiang10,17, Lan Xie1, Yuyang Jiang2, Bing Shi18,19,y, Jing Cheng1,3,4,y, Hongbing Shen12,13,y, Lin Wang10,11,12,y & Yinxue Yang5,6,y Nonsyndromic cleft lip with or without a cleft palate (NSCL/P) is among the most common human congenital birth defects and imposes a substantial physical and financial burden on affected individuals. Here, we conduct a case–control-based GWAS followed by two rounds of replication; we include six independent cohorts from China to elucidate the genetic architecture of NSCL/P in Chinese populations. Using this combined analysis, we identify a new locus at 16p13.3 associated with NSCL/P: rs8049367 between CREBBP and ADCY9 (odds ratio ¼ 0.74, P ¼ 8.98 Â 10 À 12). We confirm that the reported loci at 1q32.2, 10q25.3, 17p13.1 and 20q12 are also involved in NSCL/P development in Chinese populations. Our results provide additional evidence that the rs2235371-related haplotype at 1q32.2 could play a more important role than the previously identified causal variant rs642961 in Chinese populations. These findings provide information on the genetic basis and mechanisms of NSCL/P. 1 Department of Biomedical Engineering, Medical Systems Biology Research Center, Tsinghua University School of Medicine, Beijing 100084, China. 2 The State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China. 3 CapitalBio Corporation, Beijing 102206, China. 4 National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China. 5 General Hospital of Ningxia Medical University, Yinchuan 750004, China. 6 National Engineering Research Center for Beijing Biochip Technology, Sub-center in Ningxia, Yinchuan 750004, China. 7 Medical Genetic Center, Guangdong Women and Children Hospital, Guangzhou 511442, China. 8 Maternal and Children Metabolic-Genetic Key Laboratory, Guangdong Women and Children Hospital, Guangzhou 511442, China. 9 Biobank of Guangdong Women and Children Hospital, Guangzhou 511442, China. 10 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing 210029, China. 11 Department of Orthodontics, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing 210029, China. 12 State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, China. 13 Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing 210029, China. 14 Department of Genetic Toxicology, the Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029, China. 15 Thalassemia Diagnosis Centre, Guangdong Women and Children Hospital, Guangzhou 511442, China. 16 Department of Dentistry, Guangdong Women and Children Hospital, Guangzhou 511442, China. 17 Department of Oral and Maxillofacial Surgery, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing 210029, China. 18 The State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, China. 19 West China College of Stomatology, Sichuan University, Chengdu 610041, China. * These authors contributed equally to this work. y These authors jointly supervised this work. Correspondence and requests for materials should be addressed to Y.Y. (email: [email protected]) or to L.W. (email: [email protected]) or to H.S. (email: [email protected]). NATURE COMMUNICATIONS | 6:6414 | DOI: 10.1038/ncomms7414 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7414 onsyndromic cleft lip with or without cleft palate (NSCL/ loci at 1q32.2, 10q25.3, 17p13.1 and 20q12 are also involved in P) is among the most common human congenital birth NSCL/P development in Chinese populations. Ndefects1. Despite improvements in surgical correction techniques, this serious malformation has lifelong implications Results for health and imposes a substantial financial burden on affected Genome-wide association analysis. In the initial discovery phase, families2. Previous genetic and epidemiological studies clarified we performed a GWAS scan in two cohorts (Huaxi and Nanjing, that the aetiology of NSCL/P is multifactorial, with both genetic Supplementary Fig. 1; Supplementary Table 1). After quality and environmental factors contributing to the phenotype3–5. control (Supplementary Fig. 2), 842,556 autosomal SNPs were To date, four multi-stage, well-designed genome-wide associa- analysed (see Methods) in 858 cases and 1,248 controls (504 cases tion studies (GWAS) of NSCL/P have been conducted. In and 455 controls from the Huaxi cohort (GWAS I), and 354 cases addition to the susceptibility gene IRF6, which was previously and 793 controls from the Nanjing cohort (GWAS II)). We identified in candidate studies before the GWAS era6,7, Birnbaum performed logistic regression in each cohort with adjustments for et al.8 identified a key locus for NSCL/P at 8q24.21 using 686 sex and the significant principal components of the correspond- cases and 1,337 control subjects of European ancestry. An ing cohort to test the additive effect of minor alleles of each SNP. extension of this study, which involved 401 cases and 1,323 Meta-analysis was used to combine the results of the two cohorts controls in addition to independent replication in European trios, in the GWA scan. Because the controls in the Nanjing cohort identified two additional relevant loci at 10q25.3 and 17q22 ref. 9. were derived from imputed data from a previous study and did Beaty et al.10 applied a case–parent design to explore genome- not include newborn infants, we applied a random-effect model wide transmission patterns in 825 trios of European ancestry and in the meta-analysis when heterogeneity existed (P for Cochran’s 1,038 trios of Asian ancestry and validated the identified single- Q statistic o0.05) to avoid false-positive results caused by nucleotide polymorphisms (SNPs) with 331 Asian and 51 South putative population stratification or bias derived from age-based Asian families, 1,149 European families and 434 South/Central differences and imputation error. Quantile–quantile plots of American families, which resulted in the identification of two new genome-wide association test statistics revealed minimal inflation; susceptibility loci at 1p22.1 and 20q12. Subsequent validation of thus, substantial cryptic population substructure and differing Beaty’s study identified three other loci at 1p36.13, 3q12.3 and genotypic variants between the cases and controls were 17p13 that are significantly associated with NSCL/P11.By unlikely after meta-analysis of the two cohorts (l ¼ 1.03, combining the discovery phase of previous GWAS9,10, Ludwig Huaxi l ¼ 1.10 and l ¼ 1.06, Supplementary Fig. 3). et al.12 reported five additional loci (1p36.13, 2p21, 3p11.1, 8q21.3 Nanjing meta Thirty-four SNPs exhibited consistently significant associations and 13q31.1) that are associated with NSCL/P. These on analysis during the discovery phase (see Methods). Among observations not only targeted multiple regions influencing the these SNPs, 18 representative SNPs were selected for subsequent risk of NSCL/P but also suggested that European and Asian evaluation, and the other 16 were ignored because of their high populations may have different causal variants in the same locus linkage disequilibrium (LD) (r240.8) with at least one of the 18 as a result of different genetic backgrounds2. Although these selected SNPs (see Methods, Supplementary Table 2). Coupled studies8–10,12 were well designed, they mainly focused on with an additional 11 SNPs identified in previous studies (see susceptibility loci in people of European ancestry. Some of Methods, Table 1), we included a total of 29 SNPs in the first them involved individuals from Asia10,12, but they recruited validation conducted in Guangzhou (validation Ia), Huaxi samples from different countries and only some of them included (validation Ib) and Nanjing (validation Ic) (see Methods, Fig. 1 individuals from China. and Supplementary Fig. 4). Thus, to further elucidate the genetic architecture of NSCL/P in Chinese individuals and to identify new susceptibility loci for NSCL/P, we conduct a GWAS followed by two rounds of Replication and combined analysis. In the first round of repli- replication in a Chinese population. Using this combined cation, sixteen SNPs exhibited evidence of association with analysis, we identify a new locus at 16p13.3 associated with NSCL/P (Meta P value of validation I o0.05, Supplementary NSCL/P: rs8049367 between CREBBP and ADCY9 (odds Table 3), and we then evaluated these SNPs in the Hui population ratio ¼ 0.74, P ¼ 8.98 Â 10 À 12). We confirm that the reported (validation II), a racial minority in China. In the combined Table 1 | Twelve reported SNPs that met the validation criteria, 11 of which were included in the following validation. w z y Cytoband Published SNPs* Ref Ppublished