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.

项目摘要 使α-肌营养不良蛋白聚糖（α-DG）糖基化的基因突变是一系列肌肉萎缩的常见原因。 从先天性肌营养不良症（CMD）到儿童和成人发病的肢带型肌营养不良症 营养不良（LGMD）。这些破坏性肌病被认为是肌营养不良症并导致肌肉萎缩， 进行性无力和骨骼肌变性导致截肢， 呼吸和过早死亡。α-DG糖基转移酶基因包括FKTN，FKRP， POMT 1、POMT 2和POMGNT 1共同占遗传诊断的CMD/LGMD的50%以上。 在症状发作前或病程早期准确诊断CMD或LGMD患者 具有能够使用预防性基因治疗或其它治疗的潜力，并且在大多数情况下， 只能通过对症状前个体或产前进行基因检测。当一个新的DNA变异体出现在 在患者中观察到这些基因之一，然而，通常没有足够的证据将其归类为 致病的在这项研究中，我们将使用一种新的方法来表达和描述每一种可能的 评估基因中的错义变体，以促进我们对肌营养不良蛋白聚糖生物学的理解，改善 营养不良聚糖病基因遗传变异的解释，以及先进的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的影响的方法。 蛋白质的错义变体。此外，由于只有一部分CMD和LGMD患者具有 在已知的肌营养不良症基因的潜在致病性变异，我们将进行CRISPR筛选， 不同的细胞环境，以鉴定有助于异常α-肌营养不良蛋白聚糖功能的基因。我们的两个目标 1）定量FKTN、FKRP、POMT 1、POMT 2和POMT 3中几乎每种可能的错义变体的作用， POMGNT 1对蛋白质稳定性、α-肌营养不良蛋白聚糖糖基化和细胞粘附的影响，以及2）进行深入研究 整合多个体外测定、临床信息和 患者标本生化分析，以验证我们的DMS方法和传播致病性 预测。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 营养不良聚糖病基因变体的表征。它们还将作为一种资源， 肌肉生物学，使用遗传学改善肌营养不良聚糖病和CMD/LGMD的临床翻译 信息，并提供新的治疗方法。