Functional Analysis of Whole Exome Sequence Variants Identified in Patients with Vertebral Malformations

脊椎畸形(VM)是指发生的情况，估计发病率为 并对公众健康造成重大影响，因为它们与先天性 脊柱侧弯(CS)等情况。为了更好地了解导致 对于VM，我们建议对86例VM患者进行全外显子序列分析 通过贝勒·霍普金斯孟德尔遗传学中心和维克多·张心脏研究中心 研究所。经过严格的生物信息学分析，序列变体被确定为 远在COL11A2中。为了确定脊椎发育过程中对col11a2的需求，我们将 利用已建立的CRISPR/Cas9基因打靶方法产生斑马鱼Col11a2 功能丧失(LOF)和VM相关等位基因。观察到尾部椎体融合 在我们实验室稳定的F2col11a2 LOF突变体中，这支持了col11a2在 脊椎发育。研究其功能后果和致病性 COL11A2 R130W、R1407H和R1413H VM相关突变，我们将采用2个并行 遗传策略：(1)产生基于CRISPR/Cas9的敲入(Ki)突变；(2)挽救LOF 具有vm相关col11a2表达转基因的表型。识别基因的能力 与VM相关联将作为对VM进行分类的基础，然后可以使用 识别与模式基因突变相关的新途径、新表型，并 指导临床遗传护理患者的预防和治疗策略 更大的儿科整形外科社区。

Vertebral malformations (VMs) represent conditions that occur with an estimated incidence of 1/2000 and pose a significant public health impact due to their association with congenital scoliosis (CS) and other conditions. In order to better understand genetic variants that contribute to VM, we propose to perform whole exome sequence analysis (WES) in 86 patients with VM through the Baylor Hopkins Center for Mendelian Genetics and Victor Chang Cardiac Research Institute. Following rigorous bioinformatics analysis sequence variants have been identified so far in COL11A2. To determine the requirement for col11a2 in vertebral development, we will employ established CRISPR/Cas9 gene targeting approaches to generate zebrafish col11a2 loss-of-function (LOF) and VM-associated alleles. Caudal vertebral fusions have been observed in our laboratory in stable F2 col11a2 LOF mutants, which supports a role for col11a2 in vertebral development. To investigate the functional consequence and pathogenicity of COL11A2 R130W, R1407H, and R1413H VM-associated mutations, we will employ 2 parallel genetic strategies: (1)Create CRISPR/Cas9-based knock-in (KI) mutations; (2)Rescue LOF phenotypes with VM-associated col11a2 expression transgenes. The ability to identify genes associated with VM will serve as a foundation for classification of VM which can then be used identify novel pathways, novel phenotypes associated with patterning gene mutations, and to guide prevention and therapeutic strategies for patients cared for by the clinical genetics and greater pediatric orthopedic community.