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.

项目概要 α-、β-、γ-和 δ-肌聚糖突变会导致肌聚糖病，这是肢带型肌营养不良症的一个亚型 (LGMD) 对患者造成毁灭性影响，包括肌肉萎缩、进行性无力、肌肉退化 在症状出现前准确诊断 LGMD 患者。 或在疾病过程的早期有可能使用预防性基因疗法或其他方法 治疗，并且在大多数情况下只能通过基因检测在症状前的病例中进行。 然而，当在患者体内观察到这些基因之一出现新的 DNA 变异时，通常没有足够的证据 在这项研究中，我们将使用一种新的方法来表达和表征它。 SGCA、SGCB、SGCG 和 SGCD 基因中每一种可能的错义变异，以增进我们的理解 肌聚糖生物学，改进对 SGC 基因遗传变异的解释，并推进 LGMD 我们将采用深度突变扫描，这是一种测量大规模影响的方法。 我们将同时表达一个蛋白质的多个错义变体，其中包含所有可能的 SGC 错义变体。 培养的人类细胞中的变异，并通过利用简单但强大的特征来测量每种变异的影响 我们的两个目标是：1) 量化 SGC 基因变异。 几乎所有可能的 SGC 错义变异对 SGC 蛋白运输和膜定位的影响，以及 2) 通过整合多个SGC错义变异来预测和验证每种可能的致病性 目标 1 中的功能测定为每个变异创建致病性评分并通过确认变异 使用来自 LGMD 的 VUS 患者的组织样本进行生化预测，这些目标将揭示各自的作用。 SGC 基因中可能的错义变异会影响表达、运输、功能或与其他 SGC 的相互作用 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蛋白质。 SGC 变体的表征也将作为更好地了解肌聚糖生物学的资源。 利用遗传信息改善肌聚糖病和 LGMD 的临床转化，并为新的疾病提供信息 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