Massively-parallel functional interrogation of genetic variation in CMD-associated alpha-dystroglycan glycosylating enzymes

CMD 相关 α-肌营养不良聚糖糖基化酶遗传变异的大规模并行功能询问

• 批准号: 10802855
• 负责人: Gabriel E Haller
• 金额: $ 36.02万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-26 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10802855
• 关键词: Adult; Amino Acids; Archives; Benign; Biochemical; Biological; Biological Assay; Biology; Brain; Breathing; CRISPR screen; Caring; Cell Adhesion; Cell surface; Cells; Cellular Assay; Cessation of life; Childhood; Classification; Clinical; Clinical Data; DNA; Data; Databases; Diagnosis; Diagnostic; Disease; Dystroglycan; Enzymes; Eye; Fibroblasts; Fostering; Genes; Genetic; Genetic Code; Genetic Variation; Glycosyltransferase Gene; Goals; Human; In Vitro; Individual; International; Learning; Libraries; Limb-Girdle Muscular Dystrophies; Machine Learning; Measures; Methods; Missense Mutation; Muscle; Muscular Atrophy; Muscular Dystrophies; Mutagenesis; Mutation; Myopathy; Nucleotides; Outcome; Pathogenicity; Patients; Property; Proteins; Registries; Resolution; Resources; Saliva; Sampling; Severities; Severity of illness; Single Nucleotide Polymorphism; Skeletal Muscle; Specimen; Symptoms; Testing; Therapeutic; Time; Tissue Sample; Uncertainty; Variant; accurate diagnosis; alpha Dystroglycan; biomedical referral center; causal variant; clinical translation; congenital muscular dystrophy; disorder risk; dystroglycanopathy; fukutin; fukutin related protein; gene therapy; genetic disorder diagnosis; genetic information; genetic testing; genetic variant; glycosylation; high throughput screening; improved; in vitro Assay; machine learning algorithm; mutant; mutation screening; novel strategies; novel therapeutics; online repository; premature; prenatal; protein O-mannose beta-1,2-N-acetylglucosaminyltransferase; protein function; protein transport; protein-O-mannosyltransferase 1; protein-O-mannosyltransferase 2; segregation; tool; variant of unknown significance

PROJECT SUMMARY Mutations in genes that glycosylate alpha-dystroglycan (α-DG) are frequent causes of a spectrum of muscle disease ranging from congenital muscular dystrophy (CMD) to childhood and adult onset limb-girdle muscular dystrophy (LGMD). These devasting myopathies are deemed dystroglycanopathies and cause muscle wasting, progressive weakness, and degeneration of skeletal muscle leading to loss of ambulation, difficulties in breathing and premature death. The α-DG glycosyltransferase genes include, among others, FKTN, FKRP, POMT1, POMT2, and POMGNT1 and together account for >50% of genetically diagnosed CMD/LGMD. Accurately diagnosing patients with CMD or LGMD before symptom onset or early in the course of the disease has the potential to enable the use of preventative gene therapy or other therapeutics and in most cases can only be done through genetic testing in pre-symptomatic individuals or prenatally. When a new DNA variant in one of these genes is observed in a patient, however, there is often insufficient evidence to classify it as pathogenic. Within this study, we will use a new approach to express and characterize every possible missense variant in the assessed genes to advance our understanding of dystroglycan biology, improve the interpretation of genetic variation in dystroglycanopathy genes, and advance CMD/LGMD care and treatments. We will employ deep mutational scanning, a method for measuring the effects of massive numbers of missense variants of a protein simultaneously. Further, as only a subset of CMD and LGMD patients have potentially pathogenic variants in known muscular dystrophy genes, we will perform CRISPR screens in different cellular contexts to identify genes contributing to abnormal alpha-dystroglycan function. Our two aims are: 1) Quantifying the effect of nearly every possible missense variant in FKTN, FKRP, POMT1, POMT2, and POMGNT1 on protein stability, alpha-dystroglycan glycosylation and cellular adhesion and 2) Perform in-depth analysis of dystroglycanopathy patient variants integrating multiple in vitro assays, clinical information and patient specimen biochemical analysis to validate our DMS approach and disseminate pathogenicity predictions. The functional data we generate, the analyses we propose, and tools we build will transform the characterization of dystroglycanopathy gene variants. They will also serve as a resource to better understand muscle biology, improve the clinical translation of dystroglycanopathies and CMD/LGMD using genetic information, and inform new treatments.

项目概要 糖基化 α-肌营养不良聚糖 (α-DG) 的基因突变是一系列肌肉萎缩的常见原因 疾病范围从先天性肌营养不良症 (CMD) 到儿童和成人发病的肢带肌营养不良症 这些毁灭性的肌病被认为是营养不良症并导致肌肉萎缩， 进行性无力和骨骼肌退化，导致行走困难、行动困难 α-DG 糖基转移酶基因包括 FKTN、FKRP、 POMT1、POMT2 和 POMGNT1 合计占基因诊断 CMD/LGMD 的 50% 以上。 在症状出现前或病程早期准确诊断 CMD 或 LGMD 患者 有可能使用预防性基因疗法或其他疗法，并且在大多数情况下可以 仅当出现新的 DNA 变异时，才能通过对出现症状的个体进行基因检测来进行。 在患者中观察到其中一个基因，然而，通常没有足够的证据将其分类为 在这项研究中，我们将使用一种新的方法来表达和表征每一种可能的致病性。 所评估基因中的错义变异可促进我们对肌营养不良聚糖生物学的理解，提高 解释肌聚糖病基因的遗传变异，并推进 CMD/LGMD 护理和治疗。 我们将采用深度突变扫描，这是一种测量大量突变影响的方法。 此外，只有一部分 CMD 和 LGMD 患者同时存在蛋白质的错义变体。 已知肌营养不良症基因中的潜在致病变异，我们将在 我们的两个目标是通过不同的细胞环境来识别导致α-肌营养不良聚糖功能异常的基因。 是：1) 量化 FKTN、FKRP、POMT1、POMT2 和 POMT1 中几乎所有可能的错义变体的影响 POMGNT1 对蛋白质稳定性、α-肌营养不良聚糖糖基化和细胞粘附的影响以及 2) 进行深入研究 整合多种体外测定、临床信息和 患者样本生化分析以验证我们的 DMS 方法并传播致病性 我们生成的功能数据、我们提出的分析以及我们构建的工具将改变 它们也将作为更好地理解的资源。 肌肉生物学，利用遗传改善肌营养不良症和 CMD/LGMD 的临床转化 信息，并告知新的治疗方法。