Massively-parallel functional interrogation of genetic variation in LGMD-associated sarcoglycan genes

LGMD 相关肌聚糖基因遗传变异的大规模并行功能询问

• 批准号: 10434667
• 负责人: Gabriel E Haller
• 金额: $ 17.15万
• 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: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10434667
• 关键词: Archives; Benchmarking; Benign; Biochemical; Biocompatible Materials; Biological; Biological Assay; Biology; Breathing; Caring; Cells; Cessation of life; Characteristics; ClinVar; Clinical Trials; DNA; Data; Databases; Diagnostic; Disease; Fibroblasts; Fostering; Generations; Genes; Genetic Code; Genetic Variation; Goals; Human; In Vitro; Individual; International; Libraries; Limb-Girdle Muscular Dystrophies; Machine Learning; Measures; Membrane; Methods; Missense Mutation; Muscle; Muscular Atrophy; Mutagenesis; Mutation; Nucleotides; Pathogenicity; Patients; Property; Proteins; Registries; Resolution; Resources; Saliva; Sampling; Sarcoglycans; Single Nucleotide Polymorphism; Skeletal Muscle; Soluble Guanylate Cyclase; Symptoms; Testing; Therapeutic; Time; Tissue Sample; Tissues; Uncertainty; Validation; Variant; accurate diagnosis; base; biomedical referral center; clinical translation; delta Sarcoglycan; gamma Sarcoglycan; gene function; gene therapy; genetic information; genetic testing; genetic variant; high throughput screening; improved; in vitro Assay; mutation screening; novel strategies; novel therapeutics; premature; protein function; protein transport; tool; variant of unknown significance

PROJECT SUMMARY Mutations in α-, β-, γ-, and δ-sarcoglycan cause sarcoglyanopathies, a subset of limb-girdle muscular dystrophy (LGMD) with devastating effects for patients including muscle wasting, progressive weakness, degeneration of skeletal muscle and often premature death. Accurately diagnosing patients with 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 the majority of cases can only be done in presymptomatic cases through genetic testing. 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 SGCA, SGCB, SGCG and SGCD genes to advance our understanding of sarcoglycan biology, improve the interpretation of genetic variation in the SGC genes, and advance 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. We will express a library of all possible SGC missense variants in cultured human cells and measure the effect of each by exploiting a simple but robust characteristic of pathogenic SGC gene variants, disruption of proper protein trafficking. Our two aims are: 1) Quantifying the effect of nearly every possible SGC missense variant on SGC protein trafficking and membrane localization, and 2) Predict and validate the pathogenicity of every possible SGC missense variant by integrating multiple functional assays from Aim 1 to create a pathogenicity score for each variant and by confirming variant predictions biochemically using tissue samples from LGMD patients with VUS. These aims will reveal how each possible missense variant in SGC genes impact expression, transport, function or interaction with other SGC proteins. The functional data we generate, the analyses we propose, and tools we build will transform the characterization of SGC variants. They will also serve as a resource to better understand sarcoglycan biology, improve the clinical translation of sarcoglycanopathies and LGMD using genetic information, and inform new treatments.

项目摘要 α-、β-、γ-和δ-肌聚糖的突变引起肌聚糖病，这是肢带型肌营养不良的一个亚类 （LGMD）对患者具有破坏性影响，包括肌肉萎缩、进行性无力、 骨骼肌和经常过早死亡。在症状发作前准确诊断LGMD患者 或在疾病过程的早期具有能够使用预防性基因疗法或其他治疗的潜力。 在大多数情况下，只能通过基因检测在症状前病例中进行。 然而，当在患者中观察到这些基因中的一个新的DNA变异时， 有证据表明它是致病的在这项研究中，我们将使用一种新的方法来表达和表征 SGCA、SGCB、SGCG和SGCD基因中每一个可能的错义变体，以推进我们的理解 肌聚糖生物学，改善对SGC基因遗传变异的解释，并推进LGMD 护理和治疗。我们将采用深度突变扫描，一种测量大规模突变效应的方法。 蛋白质的错义变体的数量。我们将表达一个包含所有可能的SGC错义的库 在培养的人类细胞中的变异，并通过利用一个简单但强大的特性来测量每个变异的影响。 致病性SGC基因变异，破坏适当的蛋白质运输。我们的两个目标是：1）量化 几乎所有可能的SGC错义变体对SGC蛋白运输和膜定位的影响， 2)预测和验证每一个可能的SGC错义变异的致病性， 目的1的功能测定，以创建每个变体的致病性评分，并通过确认变体 使用来自LGMD患者VUS的组织样本进行生物化学预测。这些目标将揭示如何每个 SGC基因中可能的错义变体影响表达、转运、功能或与其他SGC的相互作用 proteins.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 SGC变体的表征。它们也将作为更好地理解肌聚糖生物学的资源， 使用遗传信息改善肌聚糖病和LGMD的临床翻译，并告知新的 治疗。

项目成果

Comprehensive functional characterization of SGCB coding variants predicts pathogenicity in limb-girdle muscular dystrophy type R4/2E.

• DOI: 10.1172/jci168156
• 发表时间: 2023-06-15
• 期刊: JOURNAL OF CLINICAL INVESTIGATION
• 影响因子: 15.9
• 作者: Li, Chengcheng;Wilborn, Jackson;Pittman, Sara;Daw, Jil;Alonso-Perez, Jorge;Diaz-Manera, Jordi;Weihl, Conrad C.;Haller, Gabe
• 通讯作者: Haller, Gabe