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

基本信息

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
项目类别：
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

项目摘要

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（牙齿发育不全、牙齿过多）、唇裂和/或唇裂板状畸形（口面裂，OFC）和唾液腺异常（异位或发育不全）是最常见的出生缺陷美国的缺陷。针对这些疾病的预防性疗法的设计需要精确的理解控制相关组织发育的转录调控网络（TRN）。研究于模型生物体的价值是无价的，例如揭示这些结构中的间充质来源于其中的神经嵴和上皮细胞主要来源于口腔外胚层。然而，尚不清楚这些 TRN 是如何在发育期间和口腔的空间域内部署。此外，这些 TRN 的各个方面可能是人类特有的，例如那些调节第二牙列发育的因素，这确实不会发生在啮齿类动物中。最后，上述所有疾病都有遗传基础，但没有一种疾病具有全部遗传基础。遗传风险得到了充分解释。了解人体组织中的 TRN 是寻找候选者的最可靠方法基因隐藏着这种风险。我们的空间转录组学 (sciSpace) 的最新进展以及捐赠的资源人类胎儿组织允许以精确的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 （受孕后 7-9、10-12、13-15 和 16-18 周）。然后我们将重点关注次要味觉和 OFC 的遗传基础。我们将使用计算算法来推断成员资格和监管调节腭上皮和腭间质不同域分化的 TRN 层次结构；顶部这些 TRN 的排名成员是拥有 OFC 缺失遗传力的有力候选者。在目标 2 中，我们将利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。我们将设计具有 2 种编码变体和 2 种非编码变体的 iPSC 与 OFC 相关，将工程 iPSC 分化为上腭细胞类型，并对分化的细胞进行实验单细胞 RNA 测序。这将揭示受影响的特定细胞类型及其发育步骤通过变异，阐明 OFC 的致病机制。这些实验将确定强口面裂遗传性缺失的候选者，改进与口面裂相关的 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。