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

表征与先天性儿科疾病相关的变异的体内平台

• 批准号: 10376759
• 负责人: Christina Gurnett
• 金额: $ 11.81万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376759
• 关键词: Affect; Alleles; Animal Model; Arthrogryposis; Biological Assay; Biological Models; Birth; Bone Tissue; CRISPR/Cas technology; Child; Childhood; Clinic; Clinical; Complex; Congenital Abnormality; Congenital clubfoot; Connective Tissue Diseases; Contracture; Country; Craniofacial Abnormalities; DNA; DNA sequencing; Defect; Development; Developmental Biology; Diagnostic; Disease; Disease Progression; Disease model; Distal; Drug Screening; Economics; Embryo; Fishes; Foundations; Funding; Genes; Genetic; Genetic Diseases; Genomics; Genotype; Goals; Human; In Vitro; Income; Joints; Knock-in; Large-Scale Sequencing; Lead; Left; Limb structure; Link; Live Birth; Longitudinal Studies; Mendelian disorder; Messenger RNA; Methods; Modeling; Molecular; Morbidity - disease rate; Morphogenesis; Morphology; Motor; Muscle; Muscle Development; Muscle Weakness; Muscle function; Musculoskeletal System; Myopathy; Myosin Heavy Chains; Nemaline Myopathies; Nonmuscle Myosin Type IIA; Operative Surgical Procedures; Organ; Pathogenicity; Patients; Pattern; Phenocopy; Phenotype; Pilot Projects; Prevalence; Research; Skeletal Muscle; Skeletal bone; Speed; Spinal Curvatures; Structural Congenital Anomalies; Supportive care; Swimming; System; Technology; Tendon structure; Testing; Therapeutic; Tissues; Tropomyosin; Universities; Variant; Washington; Work; Zebrafish; base; causal variant; cell type; clinically relevant; comparative; cost; cost effective; experience; in vivo; in vivo Model; insight; interest; limb movement; malformation; myogenesis; novel; precision medicine; programs; research clinical testing; scoliosis; screening; skeletal; small molecule; social; spine bone structure; tool; variant of unknown significance

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.

项目摘要 马蹄内翻足是世界上最常见的结构性出生缺陷之一，发病率为千分之一。 宽先天性马蹄内翻足患儿的治疗选择仅限于支持性护理，包括骨骼 支具和侵入性手术干预。如果不治疗，这在中低收入国家很常见 在缺乏专门机构管理的国家，马蹄内翻足可能导致终身的经济、社会和 身体负担。马蹄内翻足的遗传原因并不局限于一个单一的位点和致病变异 已在B3 GAT 3、CHST 14、CHST 3、COG 4、MYBPC 1、MYH 3、PITX 1和TPM 2中鉴定。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 变体仍然未被表征。 从发育的角度来看，与马蹄内翻足相关的基因控制着多种畸形的形态发生。 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。我们的首要假设是 了解并最终治疗涉及马蹄内翻足的疾病将需要体内模型， 验证变异致病性，并揭示受影响的发展计划，促进疾病。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体 (VUS)在马蹄内翻足相关疾病患者中发现，并使用致病性变体进一步建模 紊乱 远端关节挛缩（DA）是一组以挛缩为特征的相关疾病， 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，但受影响的患者往往表现出四肢以外的参与， 颅面缺损和脊柱侧凸。致病性原肌球蛋白2（TPM 2）变异体是DA的病因，但大的 已经在DA患者中鉴定了许多未表征的TPM 2变体。作为建模的切入点 DA，我们将（1）使用斑马鱼胚胎作为筛选平台，以表征TPM 2中的14种VUS，以及（2） 对三种致病性TPM 2变体进行纵向比较研究。我们希望这些证据- 原理研究将表明，肌肉发育的缺陷会导致肌肉组织的功能障碍， 肌肉骨骼系统，进而与畸形足和结构性出生缺陷相关的挛缩有关。 我们的长期目标是使用本申请中开发的方法来研究一系列基因座中的变异 与DA相关，并确定改善变异致病性的小分子。