Identification of SIX1-related genes as potential candidates for craniofacial birth defects

鉴定 SIX1 相关基因作为颅面出生缺陷的潜在候选基因

• 批准号: 9807629
• 负责人: Andre L P Tavares
• 金额: $ 15.95万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9807629
• 关键词: Address; Affect; Alleles; Applications Grants; Area; Binding; Biological Assay; Birth; Branchial arch structure; Cell Culture Techniques; Cell Line; Cephalic; Congenital Abnormality; Craniofacial Abnormalities; Data; Defect; Deformity; Disease; Dysmorphology; Ear; Embryo; Ensure; Expression Profiling; Face; Fibrinogen; First Pharyngeal Arch; Future; Gene Targeting; Genes; Genetic; Genetic Transcription; Genotype; Goals; Heterozygote; Human; Immunofluorescence Immunologic; Immunofluorescence Microscopy; In Situ Hybridization; Incisor; Individual; Jaw; Kidney; Knockout Mice; Laboratories; Luciferases; Mandible; Maxilla; Mesenchyme; Mission; Morphogenesis; Mus; Mutation; National Institute of Dental and Craniofacial Research; Neural Crest Cell; Patients; Pattern; Phenotype; Precipitation; Proteins; Reagent; Regulation; Regulator Genes; Reporter; Role; Sampling; Structure; Syndrome; Testing; Time; Tooth structure; Transcription Coactivator; Transcription Repressor/Corepressor; Work; base; craniofacial; craniofacial development; craniofacial disorder; cranium; dosage; gene interaction; hearing impairment; knock-down; middle ear; migration; neural patterning; novel; overexpression; transcription factor; transcriptome sequencing; vector

ABSTRACT Many congenital craniofacial disorders result from abnormal regulation of cranial neural crest cell (NCC) formation, migration or patterning within the mandibular portion of pharyngeal arch 1 (mandibular arch). The transcription factor SIX1 is critical for craniofacial development with mutations in SIX1 and its co-factor EYA1 identified as the underlying genetic causes of around 50% of Branchiootorenal spectrum disorder (BOS) cases. BOS is an autosomal dominant disorder, in which affected individuals that have one normal allele present variable degrees of craniofacial defects, hearing impairment, and renal abnormalities. Preliminary data from mice demonstrate that decreased levels of Six1 in heterozygotes (Six1+/-) affect the expression of four genes related to craniofacial development (Gbx2, Dlx3, Dlx2 and Hand2) more significantly than complete Six1 loss (Six1-/-); and that known (Eya1, Eya2) and putative (Pa2g4, Mcrs1, Sobp) co-factors are expressed in distinct regions of the mandibular arch with Six1. Thus, it is possible that normal dosage of Six1 regulates morphogenesis of multiple NCC-derived structures by inducing or repressing downstream genes, and that these transcriptional activities are the consequence of interactions with different levels of co-factors in distinct domains of the mandibular arch. In this R03 application I aim to identify: 1) SIX1-regulated genes within two specific mandibular arch domains; and 2) the functional interactions between SIX1 and putative co-factors PA2G4, MCRS1 and SOBP. These goals will be addressed in two specific aims. In Aim 1, genes that require proper Six1 dosage will be identified in the ventral plus intermediate domains of the mandibular arch. RNA-seq of dissected domains of the arch from Six1 heterozygotes and Six1-nulls will provide a list of genes that require normal levels of Six1. The top 10 most significantly affected genes from each genotype will be validated by quantitative real-time PCR and whole-mount and sectional RNAscope In Situ Hybridization (ISH). In Aim 2, the expression patterns of potential co-factors PA2G4, MCRS1 and SOBP will be characterized in relation to that of SIX1 in mouse embryos by dual probe whole-mount ISH and Immunofluorescence microscopy. Putative co- factors that overlap with SIX1 will be assayed in the NCC line O9-1 for whether they bind to SIX1, modulate SIX1 transcriptional activity and regulate the expression of SIX1-regulated genes from Aim 1. Results will provide a deeper understanding of SIX1 function and ultimately provide the key to understanding the underlying genetics of the remaining BOS cases or other craniofacial disorders. Validated SIX1-regulated genes and identified co-factors will be studied in future R01 applications in relation to BOS and other craniofacial disorders.

抽象的 许多先天性颅面疾病是由颅神经rest细胞（NCC）异常调节引起的 咽弓1（下颌弓）的下颌部分内的形成，迁移或图案。这 转录因子SIX1对颅面发育至关重 被确定为约50％的分支谱系谱系障碍（BOS）病例的潜在遗传原因。 BOS是一种常染色体显性疾病，其中影响有一个正常等位基因的个体 颅面缺陷，听力障碍和肾脏异常的可变程度。初步数据 小鼠表明，杂合子（六1+/-）中的六1降低会影响四个基因的表达 与完全的six1损失相比，与颅面发育（GBX2，DLX3，DLX2和HAND2）有关 （SIX1 - / - ）;以及已知的（EYA1，EYA2）和推定（PA2G4，MCRS1，SOBP）的副因素以不同的方式表达 下颌拱门的区域，带六。因此，61的正常剂量可能会调节 通过诱导或抑制下游基因的多种NCC衍生结构的形态发生，并 这些转录活性是与不同级别的共同因素相互作用的结果 下颌拱的域。在此R03应用程序中，我旨在识别：1）两个内部受调节的基因 特定的下颌拱形域； 2）SIX1与推定的副因素之间的功能相互作用 PA2G4，MCRS1和SOBP。这些目标将以两个具体目标来解决。在AIM 1中，需要的基因 将在下颌拱的腹侧和中间域中鉴定出适当的61剂量。 RNA-seq 来自六个杂合子和六个无效的拱门的解剖域将提供需要的基因列表 正常水平为六。来自每个基因型的前10个最显着影响的基因将通过 定量的实时PCR以及整个安装和截面rnascope原位杂交（ISH）。在AIM 2中 潜在的共同因素PA2G4，MCRS1和SOBP的表达模式将与此相关 通过双重探针全导量ISH和免疫荧光显微镜在小鼠胚胎中的SIX1。推定的共同 与SIX1重叠的因素将在NCC线O9-1中分析，以使其是否结合到SIX1，调节 SIX1转录活性并调节AIM 1受调节基因的表达。结果将 对Six1功能有更深入的了解，并最终为理解 其余BOS病例或其他颅面疾病的基本遗传学。经过验证的六个调节 将在未来的R01应用中研究基因和已鉴定的共同因素与BOS和其他有关 颅面疾病。