Molecular Basis of SIX2-related Frontonasal Dysplasia

额鼻发育不良（FND）是一种以眼距过远为特征的先天性颅面疾病， 前颅裂，以及影响鼻子和上唇/腭的正中面裂。这些出生 缺陷深刻地破坏了头骨和口面复合体的完整性和功能，通常导致终身 损伤FND的病因和致病机制大多未知， 显著阻碍了FND诊断和临床管理的改进。最近，三个独立的 研究已经确定了染色体2 p21处的杂合微缺失，其中SIX 2是唯一的蛋白质- 编码基因，与常染色体显性FND综合征相关，并建议SIX 2 单倍性不足是一个原因。然而，其他较小基因组缺失的杂合子患者也 含有所有SIX 2编码序列的细胞没有表现出FND。SIX 2是眼正弦同源框的成员 (SIX)家族DNA结合转录因子。在所有脊椎动物基因组中，Six 2与Six 3在物理上相连， 一个尾对尾的配置，这两个旁系同源基因组织成单独的拓扑相关 结构域（TADs）侧接一个非线性边界。而Six 2，而不是Six 3，在颅 神经嵴细胞（CNCC）来源的额鼻间充质和肾源性间充质在小鼠 胚胎发育中，Six 2-/-小鼠表现出肾脏发育不全，但正常的额鼻结构。我们的初步 研究发现，CRISPR/Cas9介导的含有Six 2基因的99-kb区域的缺失， 预测的FND边界，但不包括Six 3基因，导致严重的FND与正中面裂， 杂合子小鼠，但将缺失扩展到110-kb以包括Six 3和Six 2基因，导致 表型独特且能存活的杂合子小鼠。ENCODE染色质构象分析 从多个人类细胞系捕获的数据表明，FND相关的2 p21缺失可能破坏了 SIX 2和SIX 3之间的边界。这个R21项目将测试一个新的假设，即致病性 SIX 2相关FND的机制涉及邻近SIX 3基因的异位表达， 基因融合和增强子-启动子重新连接，而不是简单的SIX 2单倍不足，通过产生， 分析新的基因组编辑的小鼠模型以及携带不同基因的同基因多能干细胞系， FND或非FND相关的SIX 2基因组缺失。这些研究的数据有望提供新的 深入了解目前尚未表征的颅面和其他相关疾病的致病机制 结构变异改变了染色质景观，并导致分子水平的新提高， 诊断和治疗/护理这些患者。

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.