A multi-ethnic genome-wide association study identifies novel loci for non-syndromic cleft lip with or without cleft palate on 2p24.2, 17q23 and 19q13

Abstract

Orofacial clefts (OFCs), which include non-syndromic cleft lip with or without cleft palate (CL/P), are among the most common birth defects in humans, affecting approximately 1 in 700 newborns. CL/P is phenotypically heterogeneous and has a complex etiology caused by genetic and environmental factors. Previous genome-wide association studies (GWASs) have identified at least 15 risk loci for CL/P. As these loci do not account for all of the genetic variance of CL/P, we hypothesized the existence of additional risk loci. We conducted a multiethnic GWAS in 6480 participants (823 unrelated cases, 1700 unrelated controls and 1319 case-parent trios) with European, Asian, African and Central and South American ancestry. Our GWAS revealed novel associations on 2p24 near FAM49A, a gene of unknown function (P = 4.22 × 10^-8), and 19q13 near RHPN2, a gene involved in organizing the actin cytoskeleton (P = 4.17 × 10^-8). Other regions reaching genome-wide significance were 1p36 (PAX7), 1p22 (ARHGAP29), 1q32 (IRF6), 8q24 and 17p13 (NTN1), all reported in previous GWASs. Stratification by ancestry group revealed a novel association with a region on 17q23 (P = 2.92 × 10^-8) among individuals with European ancestry. This region included several promising candidates including TANC2, an oncogene required for development, and DCAF7, a scaffolding protein required for craniofacial development. In the Central and South American ancestry group, significant associations with loci previously identified in Asian or European ancestry groups reflected their admixed ancestry. In summary, we have identified novel CL/P risk loci and suggest new genes involved in craniofacial development, confirming the highly heterogeneous etiology of OFCs.

Figure 1.

Manhattan plots of –log10(P-values) from meta-analysis of case–control and TDT results in a multiethnic CL/P cohort. Results are shown for the combined multiethnic sample (A) and groups with (B) European ancestry, (C) Asian ancestry and (D) Central and South American ancestry. Colored lines denote suggestive (blue) and genome-wide (red) thresholds for significance. The genomic inflation factors, λ, are 1.088, 1.01, 0.979 and 1.045, for the multiethnic, European, Asian and Central/South American scans, respectively, indicating minor inflation in p-values consistent with the observation of multiple strongly associated loci.

Figure 2.

Regional association plots showing –log10(P-values) for genotyped (filled squares) and imputed (open circles) SNPs for novel genome-wide significant peaks. (A) Results at the 2p24 locus from the meta-analysis in the multi-ethnic cohort. (B) Results at the 19q13 locus from the meta-analysis in the multiethnic cohort. (C) Results at the 17q23 locus from meta-analysis in the European ancestry group. Plots were generated using LocusZoom (52). The recombination overlay (blue line, right y-axis) indicates the boundaries of the LD block. Points are color coded according to pairwise LD (r²) with the index SNP.

Figure 3.

Conditional analysis results for the 8q24 locus in European cases and controls. (A) Regional association plot showing –log10(P-values) for genotyped (squares) and imputed (circles) SNPs. The lead SNP in the European meta-analysis, rs72728734, is indicated by the large red diamond. (B) Regional association plot after conditioning on rs72728734. The lead SNP for the residual signal, rs184216519, is indicated by the large red diamond. The arrow indicated the located of the conditioned SNP. (C) Regional association plot after conditioning on rs184216519 and rs72728734. Arrows indicate the positions of the conditioned SNPs.