Molecular Basis of SIX2-related Frontonasal Dysplasia

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Frontonasal dysplasia (FND) are congenital craniofacial disorders characterized by ocular hypertelorism, anterior cranium bifidum, and median facial clefting affecting the nose and upper lip/palate. These birth defects profoundly disrupt the integrity and function of the skull and orofacial complex, often causing life-long impairment. The causes and pathogenic mechanisms underlying FND are mostly unknown, which significantly hinders improvement in FND diagnosis and clinical management. Recently, three independent studies have identified heterozygous microdeletions at Chromosome 2p21, in which SIX2 is the only protein- coding gene, in association with an autosomal dominant FND syndrome and suggested SIX2 haploinsufficiency as a cause. However, other patients heterozygous for smaller genomic deletions also containing all SIX2 coding sequences did not exhibit FND. SIX2 is a member of the sine oculis homeobox (SIX) family DNA-binding transcription factors. In all vertebrate genomes, Six2 is physically linked to Six3 in a tail-to-tail configuration, with these two paralogous genes organized into separate topologically associated domains (TADs) flanking a TAD boundary. Whereas Six2, but not Six3, is abundantly expressed in the cranial neural crest cell (CNCC) derived frontonasal mesenchyme and in nephrogenic mesenchyme during mouse embryogenesis, Six2-/- mice exhibited kidney hypoplasia but normal frontonasal structures. Our preliminary studies found that CRISPR/Cas9-mediated deletion of a 99-kb region containing the Six2 gene and the predicted TAD boundary, but not including the Six3 gene, caused severe FND with median facial cleft in heterozygous mice, but extending the deletion to 110-kb to include both the Six3 and Six2 genes resulted in phenotypically distinctive and viable heterozygous mice. Analysis of ENCODE chromatin conformation capture data from multiple human cell lines suggests that the FND-associated 2p21 deletions likely disrupted the TAD boundary between SIX2 and SIX3. This R21 project will test a novel hypothesis that the pathogenic mechanism of SIX2-related FND involves ectopic expression of the neighboring SIX3 gene resulting from TAD fusion and enhancer-promoter rewiring, not simply SIX2 haploinsufficiency, through generation and analyses of new genome-edited mouse model as well as isogenic pluripotent stem cell lines carrying distinct FND or non-FND associated SIX2 genomic deletions. Data from these studies are expected to provide new insights into pathogenic mechanisms of currently uncharacterized craniofacial and other disorders associated with structural variations that alter the chromatin landscape, and lead to new improvements in molecular diagnosis and treatment/care of such patients.