Genetic underpinnings of craniofacial disorders explored with spatial sequencing

通过空间测序探索颅面疾病的遗传基础

• 批准号: 10712635
• 负责人: Robert Aaron Cornell
• 金额: $ 72.79万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10712635
• 关键词: ATAC-seq; Address; Affect; Artificial Intelligence; Atlases; Candidate Disease Gene; Cell Differentiation process; Cells; Cleft Lip; Code; Computational algorithm; Conceptions; Conceptus; Congenital Abnormality; DNA; Data Set; Dental; Development; Disease; Embryo; Engineering; Epithelium; Etiology; Face; Fetus; Fishes; Gastrula; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Counseling; Goals; Growth Factor; Heritability; Heterogeneity; Human; Hypodontia; In Vitro; Individual; Inherited; Knowledge; Learning; Ligands; Linkage Disequilibrium; Location; Medial; Mesenchymal; Mesenchyme; Methods; Modeling; Morphogenesis; Multiomic Data; Mus; Mutation; Neural Crest; Newborn Infant; Oral; Oral cavity; Palate; Pathogenicity; Patients; Periderm; Preventive therapy; Protocols documentation; Resolution; Risk; Rodent; Salivary Glands; Secondary Palate; Structure; TGFB3 gene; Testing; Therapeutic Intervention; Time; Tissues; Tooth structure; Untranslated RNA; Variant; Zebrafish; cell type; craniofacial development; craniofacial disorder; design; disorder risk; effectiveness testing; experimental study; fetal; gene regulatory network; genome wide association study; human fetus tissue; human tissue; improved; induced pluripotent stem cell; loss of function; malformation; member; model organism; multiple omics; network models; oral cavity epithelium; oral ectoderm; orofacial cleft; outcome prediction; paralogous gene; permanent tooth; scaffold; single cell sequencing; single nucleus RNA-sequencing; single-cell RNA sequencing; spatiotemporal; stem cell therapy; transcription regulatory network; transcriptome sequencing; transcriptomics; virtual

Malformations of the oral cavity, which include dental anomalies (hypodontia, hyperdontia), cleft lip and or cleft plate (orofacial cleft, OFC), and salivary gland anomalies (ectopic or aplasia), are among the most common birth defects in the US. The design of preventative therapies for these disorders will require a precise understanding of the transcriptional regulatory networks (TRNs) governing development of the relevant tissues. Studies in model organisms have been invaluable, for instance revealing that mesenchyme in these structures derives from neural crest and epithelia in them derives largely from oral ectoderm. However, it is unclear how these TRNs are deployed over developmental time and within spatial domains of the mouth. Moreover, aspects of these TRNs are likely to be human specific, for instance those regulating the development of secondary dentition, which does not occur in rodents. Finally, all of the disorders mentioned above have a genetic basis, in none has all of the heritable risk been fully explained. Knowledge of the TRNs in human tissue is the surest way to find candidate genes to harbor such risk. Recent advances in our spatial transcriptomics (sciSpace), and access to donated human fetal tissue, permit these important questions to be addressed in a precise spatio-temporal manner. Here we propose, in Aim 1, to conduct sciSpace over the entire human face at four critical developmental timepoints (7-9, 10-12, 13-15, and 16-18 weeks post conception). We will then focus on the secondary palate and the genetic underpinnings of OFC. We will use computational algorithms to deduce the membership and regulatory hierarchy of TRNs regulating differentiation of distinct domains of palate epithelium and palate mesenchyme; top ranking members of these TRNs are strong candidates to harbor the missing heritability for OFC. In Aim 2, we will use the results of the first aim to develop protocols for converting induced pluripotent stem cells (iPSC) into palate epithelium and mesenchymal cells. We will engineer iPSC with 2 coding and 2 non-coding variants associated with OFC, differentiate the engineered iPSC into palate cell types, and subject the differentiated cells to single cell RNA-seq. This will reveal the specific cell types, and the step in their development, that is affected by the variants, illuminating the pathogenic mechanisms of OFC. These experiments will identify strong candidates for the missing heritability for orofacial cleft, improve functional tests of DNA variants associated with it, and provide the datasets to similarly analyze other inherited craniofacial disorders.

口腔畸形，包括牙齿异常（缺牙、多牙）、唇裂和/或裂 板（口面裂，OFC）和唾液腺异常（异位或发育不全）是最常见的出生 美国的缺陷。这些疾病的预防性治疗的设计将需要一个精确的理解 的转录调控网络（TRN）的发展有关组织。研究 模式生物是无价的，例如，揭示了这些结构中的间充质来源于 神经嵴和上皮细胞主要来源于口腔外胚层。不过，目前还不清楚这些TRN是如何 随着发育时间的推移和口腔的空间范围内展开。此外，这些TRN的方面 可能是人类特有的，例如那些调节次级牙列发育的基因， 不会发生在啮齿动物身上。最后，上述所有疾病都有遗传基础，没有一个是所有疾病都有遗传基础。 遗传风险得到充分解释。了解人体组织中的TRN是寻找候选人的最可靠方法 基因来承担这样的风险。空间转录组学（sciSpace）的最新进展，以及获得捐赠的 人类胎儿组织允许以精确时空方式解决这些重要问题。这里 在目标1中，我们建议在四个关键的发育时间点对整个人脸进行sciSpace (7-9受孕后10-12、13-15和16-18周）。然后，我们将重点放在第二腭和 OFC的遗传基础。我们将使用计算算法来推导成员资格和监管 TRN调节腭上皮和腭间充质不同区域分化的层次;顶部 这些TRN的排名成员是隐藏OFC缺失遗传力的强有力候选者。在目标2中， 将使用第一个目标的结果来开发将诱导多能干细胞（iPSC）转化为 腭上皮和间充质细胞。我们将用2个编码和2个非编码变体设计iPSC 与OFC相关，使工程化iPSC分化成腭细胞类型，并使分化的细胞 到单细胞RNA-seq。这将揭示特定的细胞类型，以及它们发育过程中受到影响的步骤 通过这些变异，阐明了OFC的致病机制。这些实验将确定强 缺失的口面裂遗传力的候选人，改善与以下相关的DNA变异的功能测试 它，并提供数据集，以类似地分析其他遗传性颅面疾病。