Expression and mutation analyses implicate ARHGAP29 as the etiologic gene for the cleft lip with or without cleft palate locus identified by genome‐wide association on chromosome 1p22

Abstract

Background Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with complex etiology reflecting the action of multiple genetic and/or environmental factors. Genome wide association studies have successfully identified five novel loci associated with NSCL/P including a locus on 1p22.1 near the ABCA4 gene. Since neither expression analysis nor mutation screening support a role for ABCA4 in NSCL/P, we investigated the adjacent gene ARHGAP29. Methods Mutation screening for ARHGAP29 protein coding exons was conducted in 180 individuals with NSCL/P and controls from the US and the Philippines. Nine exons with variants in ARHGAP29 were then screened in an independent set of 872 cases and 802 controls. Arhgap29 expression was evaluated using in situ hybridization in murine embryos. Results Sequencing of ARHGAP29 revealed eight potentially deleterious variants in cases including a frameshift and a nonsense variant. Arhgap29 showed craniofacial expression and was reduced in a mouse deficient for Irf6, a gene previously shown to have a critical role in craniofacial development. Conclusion The combination of genome wide association, rare coding sequence variants, craniofacial specific expression and interactions with IRF6 support a role for ARHGAP29 in NSCL/P and as the etiologic gene at the 1p22 GWAS locus for NSCL/P. This work suggests a novel pathway in which the IRF6 gene regulatory network interacts with the Rho pathway via ARHGAP29.

Association Studies and Direct DNA Sequencing Implicate Genetic Susceptibility Loci in the Etiology of Nonsyndromic Orofacial Clefts in Sub-Saharan African Populations

“…There is also the need to functionally validate the pathogenicity or otherwise of these variants in vivo. Rare variants in ARHGAP29 (Leslie et al 2012), PAX7 and VAX1 (Butali et al 2013;Leslie et al 2015), BMP4 (Suzuki et al 2009), FOXE1 (Moreno et al 2009), MAFB , and MSX1 (Liang et al 2012) have been observed in NSOFC cases.…”

Interferon regulatory factor 6 regulates keratinocyte migration

“…Thus, although both genes share a common function in regulating keratinocyte migration during wound healing, they appear to act in different pathways that converge at regulating the activity of RhoA. Specifically, Irf6 was shown to regulate Arhgap29 (Leslie et al, 2012), but Grhl3 was shown to regulate RhoGEF19 (also known as Arhgef19) (Caddy et al, 2010). Future studies will be needed to understand the complex gene regulatory network between these two transcription factors during keratinocyte migration and wound healing.…”

“…RhoA activation is also required for the formation of TGFb3-induced stress fibers and for mediating TGFb3 signaling during palatogenesis (Kaartinen et al, 2002). Additionally, we identified Arhgap29, a GEF with high affinity for RhoA, as a novel cleft candidate gene downstream of Irf6 (Leslie et al, 2012;Saras et al, 1997). Because Irf6 is required for proper palatogenesis (Ingraham et al, 2006;Knight et al, 2006) and is a downstream effector of TGFb3 (Knight et al, 2006;Xu et al, 2006), we hypothesize that Irf6 regulates the actin cytoskeleton in keratinocytes and alters cellular migration.…”

Genetics of Orofacial Cleft Birth Defects

“…Although no rare variants were included in functional assays, this study identified several associated clusters of rare variants near NTN1 and NOG. As with a previous candidate gene study, this targeted sequencing study revealed four nonsense mutations within ARHGAP29 [69]. All of the regions selected from GWAS showed strong statistical signals, and one of these, 17q22 near NOG, was tested for function.…”

“…Recently, there has been a resurgence of candidate gene resequencing studies following the genome-wide studies described above to provide evidence that the nearby genes are causal for OFCs. Briefly, these studies have provided evidence for GREM1 [68], MAFB [50], and ARHGAP29 [69] and have examined PAX7 [70], VAX1 [71], ABCA4 [50], and NOG [68]. Sanger sequencing has been the approach taken for these studies but is inefficient for large intervals and relies on a priori knowledge to select genes for sequencing.…”