Molecular Basis of SIX2-related Frontonasal Dysplasia

SIX2相关额鼻发育不良的分子基础

• 批准号: 10670507
• 负责人: RULANG JIANG
• 金额: $ 19.88万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670507
• 关键词: Affect; Animal Model; Anterior; CRISPR/Cas technology; Caring; Cell Differentiation process; Cell Line; Cephalic; Chromatin; Chromosomes; Clinical Management; Code; Complex; Congenital Abnormality; DNA Binding; Data; Development; Diagnosis; Disease; Disease model; Distal; Ectopic Expression; Embryonic Development; Enhancers; Etiology; Exhibits; Face; Family; Frontonasal dysplasia; Gene Deletion; Gene Expression; Gene Structure; Generations; Genes; Genetic Counseling; Genomics; Heterozygote; Homeobox; Homozygote; Human Cell Line; Impairment; Kidney; Link; Lip structure; Mediating; Mesenchyme; Molecular; Molecular Diagnosis; Molecular Genetics; Mus; Neonatal; Neural Crest Cell; Nose; Operative Surgical Procedures; Oral cavity; Orbital separation excessive; Palate; Pathogenesis; Pathogenicity; Patients; Phenotype; Pluripotent Stem Cells; Positioning Attribute; Proteins; Reproducibility; Resources; Site; Structure; Syndrome; Tail; Testing; Variant; autosome; chromosome conformation capture; craniofacial; craniofacial development; craniofacial disorder; cranium; developmental disease; genome editing; human pluripotent stem cell; improved; insight; irradiation; loss of function; member; microdeletion; midfacial hypoplasia; mouse model; mutant mouse model; novel; orofacial; paralogous gene; promoter; stem cell model; transcription factor; vertebrate genome

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.

额鼻发育不良（FND）是一种先天性颅面疾病，其特征是眼距过远， 前颅骨裂，以及影响鼻子和上唇/腭的正中面部裂隙。这些诞生 缺陷会严重破坏头骨和口面部复合体的完整性和功能，常常导致终生 损害。 FND 的病因和致病机制大多未知， 严重阻碍了 FND 诊断和临床管理的改进。最近，三个独立 研究发现染色体 2p21 存在杂合微缺失，其中 SIX2 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 SIX2 单倍体不足作为原因。然而，其他因较小基因组缺失而杂合的患者也 包含所有 SIX2 编码序列的病毒未表现出 FND。 SIX2 是正弦眼同源框的成员 (六)DNA结合转录因子家族。在所有脊椎动物基因组中，Six2 与 Six3 存在物理连锁 尾对尾的配置，这两个旁系同源基因被组织成单独的拓扑相关的 TAD 边界两侧的域 (TAD)。而 Six2（而非 Six3）在颅骨中大量表达 神经嵴细胞（CNCC）衍生的额鼻间充质和小鼠期间的肾源性间充质 在胚胎发生过程中，Six2-/- 小鼠表现出肾脏发育不全，但额鼻结构正常。我们的初步 研究发现，CRISPR/Cas9 介导的包含 Six2 基因和 99 kb 区域的删除 预测的 TAD 边界，但不包括 Six3 基因，导致严重的 FND 并伴有中面部裂 杂合子小鼠，但将缺失延伸至 110-kb 以包含 Six3 和 Six2 基因导致 表型独特且可存活的杂合小鼠。 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 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，即致病性 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-启动子重新布线，不仅仅是SIX2单倍体不足，通过生成和 分析新的基因组编辑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 SIX2 基因组缺失。这些研究的数据预计将提供新的 对目前未知的颅面和其他相关疾病的致病机制的见解 结构变化改变了染色质景观，并导致分子生物学的新改进 此类患者的诊断和治疗/护理。