Human Genetics and Clinical Translation

项目摘要 青少年特发性脊柱侧凸（AIS）是一种脊柱扭曲的情况，是最常见的儿科疾病， 肌肉骨骼疾病，影响全球3%的儿童。患有AIS的儿童有严重毁容的风险，背部 疼痛和生理功能障碍，并进行治疗，而不是预防，因为 潜在的病因是未知的。需要治疗AIS的女孩人数超过男孩五倍以上。我们 总的目的是了解AIS的生物学原因，作为早期诊断、预防和治疗AIS的一种手段。 侵入性生物治疗在此P01的支持下，我们在上一个授予期间大幅增加了 通过大规模多种族GWAS荟萃分析验证的AIS易感基因座数量。与其他项目一起 在该项目中，我们还确定了软骨细胞外基质（ECM）是AIS中的功能组织。我们 随后发现，编码ECM组分COL 11 A1和MMP 14的基因中的非同义变体是 与AIS有关。与基因组学项目3一起，我们发现了性别偏见PAX 1-COL 11 A1的证据 表情与此同时，家族中的全基因组测序确定了斑马鱼AIS家族中的候选突变， 项目2是将斑马鱼基因工程化。在这里，我们建议推动这些发现， 对AIS发病机制的理解。在该项目的一个目标中，我们将评估 通过产生Col 11 a1 fl/fl /ATC Cre系，诱导Cre重组，从软骨形成谱系中丢失Col 11 a1 在胚胎学和出生后早期的时间点。将对突变后代及其同窝仔进行评价 通过成像、定量免疫组织化学和机械强度进行表型和形态学分析 生长板和椎间盘（IVD）软骨的测试。通过项目3-基因组学，我们还将执行 单细胞RNAseq与ATAC-seq在IVD中偶联，以确定Col 11 a1在细胞水平上的作用。表征 Mmp 14在小鼠脊柱中作用，我们将描述其时空表达及其细胞特异性作用 在使用MT 1-MMPlacZ/+敲入系的脊柱发育中。在第二个目标中，我们将继续逆向工程 与项目2-斑马鱼和项目3-基因组学一起， 脊椎动物模型中脊柱畸形相关突变的后果。在互惠的方式，我们将 交叉引用项目2-斑马鱼或项目3-基因组学中鉴定的新脊柱侧凸候选等位基因， 那些在我们人类群体中发现的。在第三个目标中，我们将发现新的高风险脊柱侧凸相关变体 通过基因组测序扩展家族和AIS治疗无应答者的独特队列。我们还将 从我们在小鼠中进行的高产ENU诱导的遗传筛选中鉴定新的脊柱畸形突变体。建筑 在我们先前发现的势头下，我们将与斑马鱼项目2和基因组学合作， 项目3进一步明确脊柱发育和畸形的途径和发育机制。我们 合作还将继续定义AIS的遗传结构，并产生工具，使更好地 诊断、干预和预防。

Project Summary Adolescent idiopathic scoliosis (AIS) is a twisting condition of the spine and is the most common pediatric musculoskeletal disorder, affecting 3% of children worldwide. Children with AIS risk severe disfigurement, back pain, and physiologic dysfunction later in life, and are treated symptomatically rather than preventively because the underlying etiology is unknown. Girls requiring treatment for AIS outnumber boys by more than five-fold. Our overall purpose is to understand the biologic causes of AIS as a means to early diagnosis, prevention and non- invasive biologic treatment. With support from this P01 in the previous award period we substantially increased the number of validatedAIS susceptibility loci by large-scale multi-ethnic GWAS meta-analysis. Together with the other projects in the program we also established that the cartilage extracellular matrix (ECM) is a functional tissue in AIS. We subsequently discovered that nonsynonymous variants in genes encoding ECM components COL11A1 and MMP14 are associated with AIS. Together with genomics Project 3 we uncovered evidence of sex-biased PAX1-COL11A1 expression. In parallel, whole genome sequencing in families identified candidate mutations in AIS families that zebrafish Project 2 is engineering into orthologous zebrafish genes. Here we propose to drive these discoveries forward to develop mechanistic understanding of AIS pathogenesis. In one aim of the project, we will evaluate the consequences of Col11a1 loss from chondrogenic lineages by generating Col11a1fl/fl /ATC Cre lines, inducing cre recombination at embryologic and at early postnatal timepoints. Mutant offspring and their littermates will be evaluated phenotypically and morphologically, by imaging, quantitative immunohistochemistry and mechanical strength testing of growth plate and intervertebral disc (IVD) cartilages. With Project 3-Genomics we will also perform single cell RNAseq coupled with ATAC-seq in IVD to define the role of Col11a1 at cellular levels. To characterize the role of Mmp14 in mouse spine, we will characterize its spatio-temporal expression, and its cell-specific role in spine development using MT1-MMPlacZ/+ knockin lines. In a second aim, we will continue reverse engineering together with Project 2-Zebrafish and Project 3-Genomics to characterize the molecular and functional consequences of spine deformity-associated mutations in vertebrate models. In a reciprocal fashion we will cross-reference new scoliosis candidate alleles identified in Project 2-Zebrafish or Project 3-Genomics with those identified in our human cohorts. In a third aim we will discover novel high-risk scoliosis-associated variants by genome sequencing extended families and a unique cohort of AIS treatment non-responders. We will also identify new spine deformity mutants from our highly productive ENU-induced genetic screen in mice. Building on the momentum of our previous discoveries, we will synergize with Zebrafish Project 2 and Genomics Project 3 in further defining pathways and developmental mechanisms of spine development and deformity. Our collaborative will also continue to define the genetic architecture of AIS and yield tools that will enable better diagnosis, interventions, and preventions.