Comprehensive Characterization of Missense Variants in Factor IX

因子 IX 错义变异体的综合表征

• 批准号: 10473805
• 负责人: Nicholas Anthony Popp
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-16 至 2024-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10473805
• 关键词: Affect; Alleles; Anticoagulants; Benign; Biochemical; Biological Assay; Blood; Blood Coagulation Disorders; Blood Coagulation Factor; Cell surface; Cells; Classification; Clinical; Clinical assessments; Coagulation Process; Code; Communities; DNA sequencing; Data; Data Set; Defect; Development; Diagnosis; Disease; Engineering; F9 gene; Factor IX; Fluorescence; Functional disorder; Future; Genes; Genetic; Genetic Diseases; Genetic Variation; Genomics; Genotype; Goals; Hemophilia A; Hemophilia B; Hemorrhage; Hemostatic Agents; Hemostatic function; In Vitro; Individual; Isoantibodies; Knowledge; Length; Libraries; Life; Link; Location; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Membrane; Modeling; Modification; Outcome; Pathogenicity; Patient-Focused Outcomes; Patients; Phenotype; Physicians; Post-Translational Protein Processing; Production; Prognosis; Property; Proteins; Proxy; Recombinant Proteins; Recombinants; Recording of previous events; Replacement Therapy; Resources; Risk; Severities; Severity of illness; Single Nucleotide Polymorphism; Sorting - Cell Movement; Surface; System; Technology; Testing; Time; Transmembrane Domain; Validation; Variant; Warfarin; Work; antibody inhibitor; base; carboxylation; clinical decision-making; clinical sequencing; cohort; combat; computerized tools; disorder risk; experimental study; extracellular; genetic variant; genomic tools; high risk; high throughput screening; inhibitor; multiplex assay; mutation screening; neutralizing antibody; novel; novel strategies; predict clinical outcome; prevent; protein expression; repository; tool; variant of unknown significance

Project Summary/Abstract Most variants found in clinical DNA sequencing are classified as variants of uncertain significance (VUS), meaning these variants do not have enough information to be acted on clinically. Furthermore, of the variants that are classified as likely pathogenic or pathogenic, the clinical outcomes for patients with these variants are not often apparent from the classification alone. In particular, in hemophilia B, caused by variation in the F9 gene, it is known that the precise genetic variant is directly tied to clinical outcomes like disease severity, spontaneous bleeding risk, and risk of developing neutralizing antibodies (inhibitors) to replacement therapy. However, because of the high rate of de novo variation in this gene, many patients have novel variants for which clinical outcomes are not easily predictable. To combat this problem, this project aims to combine and utilize a set of experimental tools to characterize each of the 8,759 possible variants in F9 in high throughput with multiple functional assays, yielding a powerful dataset for reinterpreting VUS and predicting clinical outcomes in F9. As Factor IX is normally secreted from the cell, the first goal will be to generate a membrane-tethered version of Factor IX that can be displayed on the surface of mammalian cells, so that it is amenable to deep mutational scanning. This will allow me to assay key functions of Factor IX, including secretion and 𝛾- carboxylation for all possible missense variants of Factor IX. Second, I will permeabilize and assess abundance of intracellular Factor IX as a proxy for detecting null alleles which are correlated with inhibitor risk. Each of these high-throughput assays will annotate each missense variant with a quantitative score documenting the effect of that variant on the selected phenotype. Once the data are collected, the relative contribution of each Factor IX function to disease will be examined by comparing which functions are lost in variants to sequencing-based data from MyLifeOurFuture, a cohort of 1,632 patients with hemophilia B. Through this study, interpretable functional scores for thousands of Factor IX variants will be generated, potentially informing clinical decision-making. Collectively, these experiments will generate valuable data for the hemostasis community, while also establishing a new approach for scalable and accurate functional testing of variants in secreted proteins.

项目摘要/摘要 临床DNA测序中发现的大多数变异被归类为意义不确定的变异 (VUS)，这意味着这些变体没有足够的信息用于临床。此外， 被归类为可能致病或致病的变异，这些患者的临床结果 仅从分类上看，变异往往并不明显。尤其是在血友病B中，由变异引起 在F9基因中，已知精确的遗传变异与疾病等临床结果直接相关 严重程度、自发性出血风险和发展中和抗体(抑制物)以替代的风险 心理治疗。然而，由于该基因的从头变异率很高，许多患者都有新的 临床结果不容易预测的变种。为了解决这个问题，这个项目的目标是 结合并利用一组实验工具来表征F9中的每一个可能的高变种 多种功能分析的吞吐量，为重新解释VU和预测提供了强大的数据集 F9的临床结果。 由于因子IX通常是从细胞中分泌出来的，所以第一个目标将是产生一种膜系留 因子IX的一个版本，可以显示在哺乳动物细胞的表面，因此它可以适应深度 突变扫描。这将使我能够分析因子IX的关键功能，包括分泌和𝛾- 因子IX所有可能的错义变体的羧化。第二，我会渗透和评估 细胞内因子IX的丰度作为检测与抑制剂风险相关的零等位基因的替代。 这些高通量分析中的每一种都将用量化分数来注释每个错义变体 记录该变异对所选表型的影响。一旦收集到数据，相对的 每种凝血因子IX功能对疾病的贡献将通过比较哪些功能在 来自MyLifeOurFuture的基于测序的数据的变体，这是一个1632名血友病B患者的队列。 通过这项研究，将产生数千个因子IX变体的可解释功能分数， 潜在地为临床决策提供信息。总的来说，这些实验将产生有价值的数据 止血社区，同时还建立了一种可扩展和准确的功能测试的新方法 分泌蛋白质的变异体。