Association of AXIN2 with non-syndromic oral clefts in multiple populations

Abstract

We have previously shown the association of AXIN2 with oral clefts in a US population. Here, we expanded our study to explore the association of 11 AXIN2 markers in 682 cleft families from multiple populations. Alleles for each AXIN2 marker were tested for transmission distortion with clefts by means of the Family-based Association Test. We observed an association with SNP rs7224837 and all clefts in the combined populations (p = 0.001), and with SNP rs3923086 and cleft lip and palate in Asian populations (p = 0.004). We confirmed our association findings in an additional 528 cleft families from the United States (p < 0.009). We tested for gene-gene interaction between AXIN2 and additional cleft susceptibility loci. We assessed and detected Axin2 mRNA and protein expression during murine palatogenesis. In addition, we also observed co-localization of Axin2 with Irf6 proteins, particularly in the epithelium. Our results continue to support a role for AXIN2 in the etiology of human clefting. Additional studies should be performed to improve our understanding of the biological mechanisms linking AXIN2 to oral clefts.

Figure 1.

Results of TDT analyses of AXIN2 SNPs and oral clefts by population groups. (A) For all clefts. [Note strongest association for SNP rs7224837 in non-Asians (p = 0.0009) and Europeans (p = 0.005) and in the combined populations (p = 0.002). The lack of association in the other population groups indicates that the association in the combined population is primarily due to contributions from non-Asians.] (B) For CL/P (cleft lip with or without cleft palate). [Note strongest association with rs7224837 in non-Asians (p = 0.004) and in the combined populations (p = 0.001).] (C) For CLP (cleft lip with cleft palate). [Note strongest association for SNP rs3923086 in Asians and rs7224837 in non-Asians (p = 0.004) and in the combined populations (p = 0.006).]

Figure 2.

Axin2 is expressed in the mouse palate. Top, mouse embryos were sectioned coronally. (A) At E12.5, Axin2 expression (blue) is found in the palatal shelf (ps) and tissues of the developing head, including the mandible (md), tongue (t), and telencephalon (tel). (A′) Magnified view of the palatal shelves. (B) After palatal fusion at E15.5, Axin2 expression is maintained in the palate (p). (B′) Magnified view of the palate. Axin2 expression is also found in the molar tooth buds (m). Scale bars = 125 microns (bottom). Graphical representation of Axin2 and Irf6 mRNA expression levels by real-time PCR. Both gene transcripts are expressed in the murine head and palate at embryonic days (E) 12-17.

Figure 3.

Axin2 co-localizes with Irf6 in mouse palate epithelium. Coronal sections through mid- and anterior-palate regions of wild-type embryos pre- (E13, A-F; E14, G-L) and post-palate fusion (E15, M-O) were subject to immunofluorescent labeling with Axin2- and Irf6-specific polyclonal antibodies. Axin2 expression (green) is evident in palate and tooth epithelium and mesenchyme, in addition to tongue and oral epithelium and tongue musculature throughout palatogenesis (A, D, G, J, and M). Irf6 (pink) is expressed primarily in palate, tooth, oral, and tongue epithelium (B, E, H, K, and N). Merged images (yellow; C, F, I, L, and O) show co-localized expression of Axin2 and Irf6 in these epithelial cell layers critical to normal palate and tooth development.