Translating Human Height Genetics to Skeletal Biology by Functional Genomics of the Growth Plate

通过生长板的功能基因组学将人类身高遗传学转化为骨骼生物学

• 批准号: 10622603
• 负责人: Nora Edwards Renthal
• 金额: $ 17.33万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622603
• 关键词: 3-Dimensional; Affect; Automobile Driving; Biological Assay; Biology; Bone Development; Bone Growth; CD 200; CRISPR screen; Cartilage; Cell Line; Cell Maturation; Cells; Childhood; Chondrocytes; Data; Disease; Epiphysial cartilage; Family; Family member; Flow Cytometry; Future; Genes; Genetic; Genetic Determinism; Genetic Transcription; Growth; Growth Disorders; Growth and Development function; Guide RNA; Height; Human; Knock-out; Knowledge; Length; Libraries; Life Cycle Stages; Ligase; Limb structure; Link; Mediator; Mus; Pathway interactions; Patients; Phenotype; Physiologic Ossification; Physiology; Proteins; Proteome; Regulation; Research; Role; STAT1 gene; Skeletal Development; Skeleton; Structure; Sumoylation Pathway; Syndrome; Testing; Therapeutic; Transcriptional Regulation; Translating; arm; career; cell type; clinically relevant; design; experimental study; functional genomics; genome wide association study; genome wide screen; genome-wide; human data; improved; in vitro Model; inhibitor; insight; member; myocyte-specific enhancer-binding-factor 2C; novel therapeutics; premature; screening; single-cell RNA sequencing; skeletal; skeletal disorder; skeletal dysplasia; smoothened signaling pathway; transcription factor; transcriptome

Project Summary Disorders of growth plate chondrocyte maturation impact the growth of the skeleton, resulting in a spectrum of diseases from skeletal dysplasia to extreme short stature. These diverse conditions underscore the importance of the tightly regulated chondrocyte life cycle to normal epiphysial physiology, yet the genetic pathways directing chondrocyte maturation are poorly understood. The current proposal leverages an in vitro model of the growth plate to (1) conduct high-throughput, genome-wide functional knock-out (KO) screening of chondrocyte maturation, (2) prioritize screening hits with orthologues linked to human skeletal growth through genome-wide association studies (GWAS), and (3) investigate the mechanisms by which top targets act to affect chondrocyte maturation, beginning with top screening hit, Protein Inhibitor of Activated STAT1 (PIAS1). As preliminary data for the current proposal, I developed a screening assay in which a lentiviral library of 80,000 unique single-guide RNAs (sgRNAs) is transduced into Cas9+ chondrocytes to simultaneously KO 20,000 genes in replicate. This assay can robustly detect genetic determinants of chondrocyte maturation and has already identified genes highly relevant to skeletal biology, including members of the Indian hedgehog signaling family. In the present application, I intend to uncover new genetic determinants of chondrocyte maturation in the growth plate by adapting my preliminary screen to probe KOs leading to both delayed and early chondrocyte maturity and intersect these results with GWAS data from human limb length. Furthermore, I will investigate my hypothesis that PIAS1 acts to delay chondrocyte maturation through its regulation of chondrocyte transcription and protein SUMOylation, while establishing a pipeline for future mechanistic studies of top screening targets. Functional genomic screening can expedite discovery of new roles for genes previously unstudied in human growth plate chondrocytes. By identifying new functional genetic mediators of growth plate maturation, I hope to gain insight into the development of skeletal dysplasia and growth disorders while establishing targets for the design of future therapeutics.

项目摘要 生长板软骨细胞成熟障碍影响骨骼的生长，导致一系列的 从骨骼发育不良到极端矮小的疾病。这些不同的条件强调了 严格调节的软骨细胞生命周期的正常骨骺生理，但遗传途径 指导软骨细胞成熟的方法知之甚少。目前的建议利用体外模型， 所述生长板用于（1）进行高通量、全基因组功能性敲除（KO）筛选， 软骨细胞成熟，（2）优先筛选与人类骨骼生长相关的直系同源物， 全基因组关联研究（GWAS）;（3）研究顶级靶点作用于 影响软骨细胞成熟，从最高筛选命中开始，激活的STAT 1蛋白抑制剂（PIAS 1）。 作为当前提案的初步数据，我开发了一种筛选测定，其中慢病毒文库 将80，000个独特的单向导RNA（sgRNA）转导到Cas9+软骨细胞中，同时KO 两万个基因复制。该测定法可以稳健地检测软骨细胞成熟的遗传决定因子， 已经鉴定出与骨骼生物学高度相关的基因，包括印度刺猬的基因 信号家族在本申请中，我打算揭示软骨细胞的新的遗传决定因素， 通过调整我的初步筛选来探测科斯， 早期软骨细胞成熟，并将这些结果与人类肢体长度的GWAS数据交叉。而且我 我将研究我的假设，PIAS 1通过调节软骨细胞的生长来延迟软骨细胞的成熟。 软骨细胞转录和蛋白质SUMO化，同时为未来的机制研究建立管道 筛选的最佳目标功能基因组筛选可以加速发现基因的新作用 以前未在人类生长板软骨细胞中研究过。通过鉴定新的功能性遗传介质， 生长板成熟，我希望能够深入了解骨骼发育不良和生长障碍的发展 同时为未来的治疗设计建立目标。

项目成果

Genome-wide CRISPR screening of chondrocyte maturation newly implicates genes in skeletal growth and height-associated GWAS loci.

• DOI: 10.1016/j.xgen.2023.100299
• 发表时间: 2023-05-10
• 期刊: Cell genomics
• 作者: Baronas JM;Bartell E;Eliasen A;Doench JG;Yengo L;Vedantam S;Marouli E;GIANT Consortium;Kronenberg HM;Hirschhorn JN;Renthal NE
• 通讯作者: Renthal NE

Genetics of skeletal proportions in two different populations.

两个不同人群骨骼比例的遗传学。

• DOI: 10.1101/2023.05.22.541772
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Bartell,Eric;Lin,Kuang;Tsuo,Kristin;Gan,Wei;Vedantam,Sailaja;Cole,JoanneB;Baronas,JohnM;Yengo,Loic;Marouli,Eirini;Amariuta,Tiffany;Chen,Zhengming;Li,Liming;GIANTconsortium;ChinaKadoorieBiobankCollaborativeGroup;Renthal,No
• 通讯作者: Renthal,No