An in vivo platform to characterize variants associated with congenital pediatric disorders

项目总结 马蹄内翻足是最常见的结构性出生缺陷之一，全球活产儿发病率为千分之一。 很宽。先天性马蹄内翻足儿童的治疗选择仅限于支持性护理，包括骨骼护理 支撑和侵入性外科干预。如果不治疗，这在中低收入人群中很常见 缺乏专门机构管理的国家，马蹄内翻足可能导致终生的经济、社会和 身体负担。马蹄内翻足的遗传原因并不局限于单一的基因座和致病变异。 已在B3GAT3、CHST14、CHST3、COG4、MYBPC1、MYH3、PITX1和TPM2中发现。大规模 测序计划已经在马蹄内翻足相关基因座中发现了无数的变异，但其中绝大多数 变种仍未确定其特征。 从发育的角度来看，与马蹄内翻足相关的基因控制着多个 包括骨骼、肌肉和结缔组织在内的系统，以及导致马蹄内翻足的疾病机制 可能涉及多个器官之间不完整或不适当的相互作用。我们最重要的假设是 了解并最终治疗与马蹄内翻足有关的疾病将需要能够 验证变异的致病性，并发现促进疾病的受影响的发育程序。通过 利用斑马鱼遗传学的速度和多功能性，我们将筛选未知意义的变异 在马蹄内翻足相关疾病患者中发现(VUS)，并使用致病变种进一步建模 精神错乱。 远端关节紊乱(DA)是一组以肌挛缩为特征的相关疾病，其定义为 作为肌肉和关节的永久性缩短，抑制肢体运动和功能。马蹄内翻足就是其中之一 DA的诊断特征，但受影响的患者通常表现为四肢以外的病变，包括 头面部缺陷和脊柱侧弯。致病原肌球蛋白2(TPM2)变异是DA的病因，但 已经在DA患者中发现了一些未表征的TPM2变异。作为建模的入口点 DA，我们将(1)使用斑马鱼胚胎作为筛选平台来表征TPM2中的14个VU，以及(2) 对三种致病TPM2变异体进行纵向比较研究。我们希望这些证据- 原理研究将表明，肌肉发育缺陷会导致肌肉组装功能障碍 肌肉骨骼系统，进而导致与马蹄内翻足和结构性出生缺陷相关的痉挛。 我们的长期目标是使用在此应用程序中开发的方法来研究一系列基因座的变异 与DA相关，并鉴定改善变异致病性的小分子。

PROJECT SUMMARY Clubfoot is one of the most common structural birth defects with a prevalence of 1 in 1000 live births world- wide. Therapeutic options for children born with clubfoot are limited to supportive care including skeletal bracing and invasive surgical interventions. If left untreated, which is common in low- to middle- income countries that lack specialized institutional management, clubfoot can incur lifelong economic, social, and physical burdens. The genetic causes of clubfoot are not confined to a single locus and pathogenic variants have been identified in B3GAT3, CHST14, CHST3, COG4, MYBPC1, MYH3, PITX1, and TPM2. Large-scale sequencing initiatives have identified countless variants in clubfoot-associated loci, but a vast majority of these variants remain uncharacterized. From a developmental standpoint, the genes associated with clubfoot control the morphogenesis of multiple systems including bone, muscle, and connective tissues, and the disease mechanisms contributing to clubfoot likely involve incomplete or inappropriate interactions among multiple organs. Our overarching hypothesis is that understanding and ultimately treating disorders involving clubfoot will require in vivo models that can validate variant pathogenicity and uncover the affected developmental programs that promote disease. By leveraging the speed and versatility of zebrafish genetics, we will screen variants of unknown significance (VUS) identified in patients with clubfoot-associated disorders and use pathogenic variants to further model disorders. Distal Arthrogryposes (DA) are a group of related disorders characterized by contractures, which are defined as a permanent shortening of muscles and joints that inhibit limb movement and function. Clubfoot is one diagnostic feature of DA, but affected patients often show involvement outside the extremities including craniofacial defects and scoliosis. Pathogenic Tropomyosin 2 (TPM2) variants are causative of DA, but a large number of uncharacterized TPM2 variants have been identified in DA patients. As an entry point to modeling DA, we will (1) use zebrafish embryos as a screening platform to characterize fourteen VUS in TPM2, and (2) perform comparative longitudinal studies of three pathogenic TPM2 variants. We expect that these proof-of- principle studies will show that defects in muscle development lead to dysfunctional assembly of the musculoskeletal system, and in turn to the contractures associated with clubfoot and structural birth defects. Our long-term goals are to use the methods developed in this application to study variants in an array of loci associated with DA, and to identify small molecules that ameliorate variant pathogenicity.