Comprehensive Characterization of Missense Variants in Factor IX

因子 IX 错义变异体的综合表征

• 批准号: 10467979
• 负责人: Nicholas Anthony Popp
• 金额: $ 5.1万
• 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/10467979
• 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; Structure; 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/antagonist; 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.

项目总结/摘要 临床DNA测序中发现的大多数变异都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可以在临床上采取行动。此外， 分类为可能致病或致病的变异，这些患者的临床结果 仅从分类来看，变异往往并不明显。特别是在血友病B中，由变异 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、自发性出血风险和产生中和抗体（抑制剂）的风险 疗法然而，由于该基因的高从头变异率，许多患者具有新的遗传缺陷。 临床结果不容易预测的变异。为了解决这一问题，该项目旨在 联合收割机并利用一组实验工具来表征F9中的8，759种可能变体中的每一种， 多功能检测的通量，产生一个强大的数据集，用于重新解释VUS和预测 F9的临床结果。 由于因子IX通常从细胞中分泌，因此第一个目标将是产生膜系连的 因子IX的版本，可以展示在哺乳动物细胞的表面上，因此它适合于深入研究。 突变扫描这将使我能够分析因子IX的关键功能，包括分泌和分泌。 针对因子IX的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无效等位基因的代表。 这些高通量检测中的每一个都将用定量评分来注释每个错义变体 记录该变体对所选表型的影响。收集数据后，相对 将通过比较哪些功能在疾病中丢失来检查每个因子IX功能对疾病的贡献。 来自MyLifeOurFuture的基于测序的数据的变体，MyLifeOurFuture是一个包含1，632名血友病B患者的队列。 通过这项研究，将产生数千种因子IX变体的可解释功能评分， 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 分泌蛋白质的变体。