Molecular Basis of SIX2-related Frontonasal Dysplasia

额鼻发育不良(FND)是一种先天性颅面部疾病，以眼部过度调节为特征， 前颅骨二裂，影响鼻子和上唇/腭部的正中面部裂隙。这些出生 缺陷会严重破坏颅骨和口面部复合体的完整性和功能，通常会导致终生 减损。FND的病因和致病机制大多尚不清楚， 严重阻碍了FND诊断和临床管理的改进。最近，三个独立的 研究已经发现了染色体2p21上的杂合性微缺失，其中SIX2是唯一的蛋白质- 编码基因与常染色体显性遗传性FND综合征和提示的SIX2相关 单倍体功能不全是一个原因。然而，其他杂合子较小的基因组缺失的患者也 包含所有SIX2编码序列的序列没有表现出FND。SIX2是Sine Oculis Homeobox的成员 (六)家族DNA结合转录因子。在所有脊椎动物基因组中，SIX2与SIX3在物理上是连锁的 尾巴到尾巴的构型，这两个相似的基因被组织成单独的拓扑关联 位于TAD边界两侧的域(TADS)。然而，SIX2，而不是SIX3，在颅骨中大量表达 小鼠额鼻间充质和肾源性间充质中的神经脊细胞 在胚胎发育过程中，SIX2-/-小鼠表现出肾脏发育不良，但额鼻结构正常。我们的预赛 研究发现，CRISPR/Cas9介导的含有SIX2基因的99kb区域的缺失和 预测的TAD边界，但不包括Six3基因，导致严重的FND伴正中面部裂 杂合子小鼠，但将缺失扩大到110-kb，包括Six3和SIX2基因，导致 表型独特且可行的杂合子小鼠。编码区染色质构象分析 从多个人类细胞系捕获的数据表明，与FND相关的2p21缺失可能被破坏 SIX2和SIX3之间的TAD边界。这个R21项目将测试一个新的假设，即致病因子 SIX2相关FND的机制涉及邻近Six3基因的异位表达 TAD融合和增强子-启动子重新连接，而不仅仅是SIX2单倍体不足，通过生成和 新的基因组编辑小鼠模型以及携带不同基因的同源多能干细胞系的分析 FND或非FND相关的SIX2基因组缺失。来自这些研究的数据预计将提供新的 对目前尚不典型的颅面及其他相关疾病的致病机制的见解 具有改变染色质景观的结构变化，并导致分子的新改进 此类患者的诊断和治疗/护理。

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.