A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4

Abstract

Case-parent trios were used in a genome-wide association study of cleft lip with and without cleft palate. SNPs near two genes not previously associated with cleft lip with and without cleft palate (MAFB, most significant SNP rs13041247, with odds ratio (OR) per minor allele = 0.704, 95% CI 0.635-0.778, P = 1.44 x 10(-11); and ABCA4, most significant SNP rs560426, with OR = 1.432, 95% CI 1.292-1.587, P = 5.01 x 10(-12)) and two previously identified regions (at chromosome 8q24 and IRF6) attained genome-wide significance. Stratifying trios into European and Asian ancestry groups revealed differences in statistical significance, although estimated effect sizes remained similar. Replication studies from several populations showed confirming evidence, with families of European ancestry giving stronger evidence for markers in 8q24, whereas Asian families showed stronger evidence for association with MAFB and ABCA4. Expression studies support a role for MAFB in palatal development.

Figure 1

Manhattan plots of log₁₀(p-values) from transmission disequilibrium test (TDT) for autosomal SNPs on CL/P case-parent trios (omitting SNPs flagged for QC). (a) Results based on all 1908 CL/P trios; (b) Results based on 825 CL/P case-parent trios of European ancestry; (c) Results based on 1038 CL/P case-parent trios of Asian ancestry.

Figure 2

Significance and effect size for SNPs near MAFB based on all CL/P trios. a) −log₁₀(p-value) for allelic TDT for 17 SNPs near MAFB on chr. 20q11; b) Estimated OR(case) from a conditional logistic regression and their 95%CI under an additive model; c) Physical position of tested SNPs and the single exon of MAFB.

Figure 3

Significance and effect size for SNPs in and near ABCA4 based on all CL/P trios. a) −log₁₀(p-value) for allelic TDT for 98 SNPs in or near ABCA4 on chr. 1p22.1; b) Estimated OR(case) from a conditional logistic regression and their 95%CI under an additive model fit to 1908 CL/P case-parent trios; c) Physical position of tested SNPs and combined exons of the ABCA4 gene.

Figure 4

Mafb, and not Abca4, is expressed during the development of the secondary palate in the mouse. In situ hybridization for Mafb on whole mount e13.5 embryos (a–d) shows expression in craniofacial ectoderm, vibrissae, and neural-crest derived mesoderm in murine embryos. Signal was also detected in the elevated palatal shelves (b – view of the roof of the mouth). Immunofluorescence staining for Mafb (red) on e13.5 palatal sections shows Mafb localized in the epithelium of the palatal shelves (f) and in the medial edge epithelium during palatal fusion on e14.5 tissue sections (g, h). Expression is also detected at the base of the nasal septum and in the tongue epithelium (g). Note the absence of signal in the sense probe (b, d) and no primary control (e). Immunofluorescence staining for Abca4 (green) on adult murine retina (i) and e14.5 palatal sections (j) show the presence of Abca4 in the rim of rods photoreceptor cells of the retina and its absence in orofacial structures. Nuclei were counterstained with DAPI (blue). v, vibrissae; p, palatal shelf; t, tongue, ns, nasal septum. (Scale bar = 100 μm panels e–h; = 50 μm panel i).