FOXE1 association with both isolated cleft lip with or without cleft palate, and isolated cleft palate

Abstract

Nonsyndromic orofacial clefts are a common complex birth defect caused by genetic and environmental factors and/or their interactions. A previous genome-wide linkage scan discovered a novel locus for cleft lip with or without cleft palate (CL/P) at 9q22-q33. To identify the etiologic gene, we undertook an iterative and complementary fine mapping strategy using family-based CL/P samples from Colombia, USA and the Philippines. Candidate genes within 9q22-q33 were sequenced, revealing 32 new variants. Concurrently, 397 SNPs spanning the 9q22-q33 2-LOD-unit interval were tested for association. Significant SNP and haplotype association signals (P = 1.45E - 08) narrowed the interval to a 200 kb region containing: FOXE1, C9ORF156 and HEMGN. Association results were replicated in CL/P families of European descent and when all populations were combined the two most associated SNPs, rs3758249 (P = 5.01E - 13) and rs4460498 (P = 6.51E - 12), were located inside a 70 kb high linkage disequilibrium block containing FOXE1. Association signals for Caucasians and Asians clustered 5' and 3' of FOXE1, respectively. Isolated cleft palate (CP) was also associated, indicating that FOXE1 plays a role in two phenotypes thought to be genetically distinct. Foxe1 expression was found in the epithelium undergoing fusion between the medial nasal and maxillary processes. Mutation screens of FOXE1 identified two family-specific missense mutations at highly conserved amino acids. These data indicate that FOXE1 is a major gene for CL/P and provides new insights for improved counseling and genetic interaction studies.

Figure 1.

Maximum summed multipoint HLODs on chromosome 9 under a dominant model obtained from the 10 cM genome scan of CL/P in the CIDR-7 studies. Each population's contribution to the total HLOD is detailed elsewhere (11,68).

Figure 2.

FBAT association results. (A) 331 Illumina markers including the 2LOD interval. (B) Candidate genes interval between ROR2-ALDOB (178 SNPs). (C) 357 kb critical region (34 SNPs). The size of the circles represents the over-transmission of the common (large circles) or rare allele (small circles).

Figure 3.

LD Blocks for Stage 3—357 kb critical region SNPs. FBAT association results −log₁₀(P-value) are displayed by upper track. (A) Colombia, (B) the USA, (C) the Philippines.

Figure 3.

LD Blocks for Stage 3—357 kb critical region SNPs. FBAT association results −log₁₀(P-value) are displayed by upper track. (A) Colombia, (B) the USA, (C) the Philippines.

Figure 4.

Haplotype window analysis results for Stage 3—357 kb critical region. Rectangles represent the size of the haplotype according to SNPs involved. Gray scale represent the −log₁₀(P-value) results. (A) Colombia (B) USA (C) Philippines (D) Combined. See Supplementary Material, Appendix Table S1 for allele correspondence to specific nucleotides.

Figure 5.

FBAT results. (A) All cleft phenotypes. (B) CLO by population. (C) CLPO by population. (D) CL/P by population and (E) CP by population.

Figure 5.

FBAT results. (A) All cleft phenotypes. (B) CLO by population. (C) CLPO by population. (D) CL/P by population and (E) CP by population.

Figure 6.

Over-transmission of the associated allele. (A) CL/P and (B) CLPO.

Figure 7.

Haplotype window analysis results for replication panel markers and the CL/P phenotype. Rectangles represent the size of the haplotype according to SNPs involved. Colors represent the −log₁₀(P-value) results. i_rs#, markers typed by CIDR; rs#_h, markers genotyped in our laboratories. See Supplementary Material, Appendix Table S11 for allele correspondence to specific nucleotides.

Figure 8.

CL/P% maternal (MT) versus paternal (PT). Difference values above 10 or below −10 are considered significant at an α of 0.05. See Supplementary Material, Appendix Table S11 for allele correspondence to specific nucleotides and transmission direction of each allele.

Figure 9.

Foxe1 expression at 45 somites. (A) Whole mount in situ. (B) Section in situ. Expression in caudal epithelium of medial nasal and maxillary processes.