61.09 Using Small Molecule Screens to Identify New IRF6 Pathways and Mitigate Orofacial Cleft Pathogenesis

Posted on January 26, 2017

E. Li1,2,3, B. Garrity1,2,3, D. Truong1,2,3, K. Mukherjee1,2,3, E. C. Liao1,2,3  1Massachusetts General Hospital,Boston, MA, USA 2Harvard Medical School,Boston, MA, USA 3Harvard Stem Cell Institute,Cambridge, MA, USA

Introduction:

Mutations in the transcription factor IRF6 represent the most common genetic determinant of both syndromic and nonsyndromic cleft lip and/or palate (CL/P). We hypothesized that the IRF6 gene regulatory network contains pharmacological targets that could prevent CL/P in utero, much like the dramatic effect of prenatal folate supplementation on decreasing the incidence of spina bifida.

CRISPR genome editing was used to disrupt irf6 in zebrafish. All mutant embryos displayed an embryonic epithelium (periderm) rupture phenotype, which represents a sensitive platform for high-throughput chemical screening to identify small molecules that could modulate irf6 regulatory pathways. Using our irf6 mutant model, we present a screen of known bioactive compounds for modulation of Irf6 activity in zebrafish, and identification of pathways that could play a role in palate development.

Methods:

Mutant irf6-/- embryos were dispensed 10 embryos/well in 96-well plates and incubated in media containing propidium iodide (renders ruptured embryos fluorescent). The ICCB known-bioactives library consisting of 480 FDA-approved drugs with well-characterized biological targets was screened. Mutant embryos treated with DMSO were used as solvent controls. Wildtype embryos treated with drugs were used as toxicity controls. Timelapse images of the wells were captured by automated brightfield and fluorescence microscopy and analyzed with ImageJ software. Molecular pathways associated with the positive hits were identified through the library index and analyzed using computational modeling programs.

Results:

65 of the 480 small molecules screened reached statistical significance in delaying periderm rupture compared to DMSO-treated controls without causing developmental delays in wildtype embryos. The molecular targets of the small molecule hits were analyzed by Gene Ontology and revealed not only molecular pathways previously known to play crucial roles in palate development such as PDGF and FGF, but also novel pathway connections between IRF6 and retinoic acid, aryl hydrocarbon, and adenosine pathways among others. Furthermore, when these pathways were aberrantly modulated in wildtype zebrafish embryos, craniofacial defects were observed.

Conclusion:

Zebrafish irf6-/- embryos represent a robust platform for high-throughput small molecule screens to identify modulators of IRF6 capable of potentially mitigating cleft pathogenesis. The results identified several critical developmental pathways, some previously reported as essential in palate development, while others are not yet characterized. These novel pathways could represent unexplored regulatory mechanisms of palate development and novel nodes of pharmacological intervention for orofacial clefting.

20.01 Using Small Molecule Screens to Identify New IRF6 Pathways and Mitigate Orofacial Cleft Pathogenesis

Posted on January 25, 2017

E. Li1,2,3, B. Garrity1,2,3, D. Truong1,2,3, K. Mukherjee1,2,3, E. C. Liao1,2,3  1Massachusetts General Hospital,Boston, MA, USA 2Harvard Medical School,Boston, MA, USA 3Harvard Stem Cell Institute,Cambridge, MA, USA

Introduction:

Mutations in the transcription factor IRF6 represent the most common genetic determinant of both syndromic and nonsyndromic cleft lip and/or palate (CL/P). We hypothesized that the IRF6 gene regulatory network contains pharmacological targets that could prevent CL/P in utero, much like the dramatic effect of prenatal folate supplementation on decreasing the incidence of spina bifida.

CRISPR genome editing was used to disrupt irf6 in zebrafish. All mutant embryos displayed an embryonic epithelium (periderm) rupture phenotype, which represents a sensitive platform for high-throughput chemical screening to identify small molecules that could modulate irf6 regulatory pathways. Using our irf6 mutant model, we present a screen of known bioactive compounds for modulation of Irf6 activity in zebrafish, and identification of pathways that could play a role in palate development.

Methods:

Mutant irf6-/- embryos were dispensed 10 embryos/well in 96-well plates and incubated in media containing propidium iodide (renders ruptured embryos fluorescent). The ICCB known-bioactives library consisting of 480 FDA-approved drugs with well-characterized biological targets was screened. Mutant embryos treated with DMSO were used as solvent controls. Wildtype embryos treated with drugs were used as toxicity controls. Timelapse images of the wells were captured by automated brightfield and fluorescence microscopy and analyzed with ImageJ software. Molecular pathways associated with the positive hits were identified through the library index and analyzed using computational modeling programs.

Results:

65 of the 480 small molecules screened reached statistical significance in delaying periderm rupture compared to DMSO-treated controls without causing developmental delays in wildtype embryos. The molecular targets of the small molecule hits were analyzed by Gene Ontology and revealed not only molecular pathways previously known to play crucial roles in palate development such as PDGF and FGF, but also novel pathway connections between IRF6 and retinoic acid, aryl hydrocarbon, and adenosine pathways among others. Furthermore, when these pathways were aberrantly modulated in wildtype zebrafish embryos, craniofacial defects were observed.

Conclusion:

Zebrafish irf6-/- embryos represent a robust platform for high-throughput small molecule screens to identify modulators of IRF6 capable of potentially mitigating cleft pathogenesis. The results identified several critical developmental pathways, some previously reported as essential in palate development, while others are not yet characterized. These novel pathways could represent unexplored regulatory mechanisms of palate development and novel nodes of pharmacological intervention for orofacial clefting.

19.02 Personalized Medicine in Cleft Lip and Palate: Functional Genomics Analysis of IRF6 Gene Variants

Posted on January 25, 2017

E. Li1,2,3, B. Garrity1,2,3, D. Truong1,2,3, K. Mukherjee1,2,3, E. C. Liao1,2,3  1Massachusetts General Hospital,Boston, MA, USA 2Harvard Medical School,Boston, MA, USA 3Harvard Stem Cell Institute,Cambridge, MA, USA

Introduction:

Cleft lip and/or palates (CL/P) are among the most common congenital malformations. Mutations in the transcription factor gene IRF6 represent the most significant genetic contributors to orofacial clefts. Hundreds of novel sequence variations in IRF6 have been reported. However, the functional significances of the myriad of IRF6 mutations remain difficult to ascertain as in silico prediction programs have poor predictive power and often provide conflicting results. To address this gap in knowledge, we hypothesized that the protein function of IRF6 gene variants could be rapidly assessed by their ability to rescue the irf6-/- mutant phenotype in animal models.

Methods:

CRISPR genome editing was used to generate irf6-/- zebrafish. Previously uncharacterized IRF6 variants were identified and categorized by computational predictions from Polyphen2 and SIFT. Variants were introduced to wildtype irf6 cDNA by site-directed mutagenesis; mRNA was produced by in vitro transcription and microinjected into irf6-/- embryos to assess for phenotypic and functional rescue.

Results:

All offspring of irf6-/- females displayed an embryonic rupture phenotype caused by defects in the embryonic periderm, a simple squamous epithelium surrounding the embryo that models the human oral epithelium. The periderm rupture model is validated for use in the analysis of human IRF6 function, as injection of human IRF6 mRNA into mutant zebrafish embryos can fully rescue the mutant phenotype. Human IRF6 gene variant mRNAs were then injected in a rapid and accurate rescue assay to assess the effects of human IRF6 mutations on protein function.

The IRF6 gene variant rescue results revealed that neither location of the mutations within the IRF6 protein nor computational programs were accurate predictors of whether observed human IRF6 mutations actually resulted in defective proteins. Several variants predicted to be benign failed to rescue, while others predicted to be deleterious by both computational programs were able to rescue not only periderm rupture, but also normal craniofacial development.

Conclusion:

The zebrafish irf6-/- rescue assay rapidly addressed the functional significance of novel IRF6 mutations and overcame computational limitations for predicting variant pathogenicity. These validated biological results can help improve future computational predictions, and moreover, unlock the possibility for rapidly characterizing yet undiscovered IRF6 variants from orofacial cleft patients, illustrating a generalizable functional genomics paradigm of personalized medicine.