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 糖基化α-Dystroglycan（α-DG）的基因中的突变通常是肌肉谱的原因 从先天性肌肉营养不良（CMD）到儿童和成人发作肢体肌肉的疾病 营养不良（LGMD）。这些破坏性的肌病被认为是营养不良的肿瘤病，会导致肌肉浪费， 进行性无力和骨骼肌的变性导致卧床丧失，困难 呼吸和过早死亡。 α-DG糖基转移酶基因包括FKTN，FKRP， POMT1，POMT2和POMGNT1占遗传诊断为CMD/LGMD的50％。 在症状发作之前或疾病早期，准确诊断患有CMD或LGMD的患者 有潜力能够使用预防基因疗法或其他疗法，在大多数情况下 仅通过在症状前或产前进行基因检测来完成。当新的DNA变体中 但是，在患者中观察到这些基因之一，但是，通常没有足够的证据将其归类为 致病性。在这项研究中，我们将使用一种新方法来表达和表征一切可能 评估基因中的错义变体，以提高我们对多根糖生物学的理解，改善 遗传疾病基因的遗传变异的解释，并推进CMD/LGMD护理和治疗。 我们将采用深层突变扫描，一种测量大量效果的方法 蛋白质的错义变体。此外，由于只有一部分CMD和LGMD患者具有 在已知肌肉营养不良基因的潜在致病性变异，我们将在 不同的细胞环境，以识别有助于异常α-二粘膜功能的基因。我们的两个目标 是：1）量化FKTN，FKRP，POMT1，POMT2和 POMGNT1关于蛋白质稳定性，α-毛线糖基化糖基化和细胞粘合剂的蛋白质稳定性和2）深入进行 分析肿瘤疾病患者变体，这些变体整合了多个体外测定，临床信息和 患者标本生化分析以验证我们的DMS方法并传播致病性 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 多糖基因疾病基因变体的表征。他们还将作为更好地理解的资源 肌肉生物学，改善使用遗传的临床症状转化和CMD/LGMD的临床翻译 信息，并告知新的治疗方法。