Resolving Uncertainty in Alagille Syndrome Diagnostics

解决阿拉吉尔综合征诊断中的不确定性

• 批准号: 10734881
• 负责人: Nancy Bettina Spinner
• 金额: $ 58.15万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10734881
• 关键词: Accounting; Affect; Alagille Syndrome; Atlases; Benign; Biological Assay; Calibration; Cell Line; Cells; Childhood; Classification; ClinVar; Clinical; Complex; DNA; Data; Detection; Development; Diagnosis; Diagnostic; Disease; Etiology; Evaluation; Exons; Eye; Face; Facies; Formalin; Future; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic; Genetic Diseases; Genetic Variation; Genomic medicine; Genomics; Genotype; Heart; High Prevalence; Individual; Kidney; Knowledge; Libraries; Ligands; Liver; Liver diseases; Managed Care; Measures; Membrane; Mendelian disorder; Methodology; Methods; Missense Mutation; Modeling; Molecular; Mutation; Notch Signaling Pathway; Nucleotides; Paraffin Embedding; Pathogenicity; Patient Care; Patients; Persons; Phenotype; Positioning Attribute; Property; Protein Truncation; Proteins; Proteomics; Receptor Signaling; Reporter; Reporting; Resolution; Sampling; Series; Severity of illness; Signal Transduction; Site; System; Techniques; Testing; Therapeutic; Tissue Sample; Uncertainty; Validation; Variant; Work; autosome; clinical diagnostics; design; disorder control; experimental study; glycosylation; high throughput screening; improved; innovation; liver transplantation; loss of function; multiplex assay; mutant; notch protein; protein function; receptor; sample fixation; spine bone structure; tool; transcriptomics; variant of unknown significance

PROJECT SUMMARY/ABSTRACT Uncertainty in genomic diagnostics creates a barrier to realizing the full potential of genomic medicine. Uncertainty is evident in: 1) our inability to determine if some DNA variants are pathogenic or benign and 2) our inability to predict to what extent a person with a disease-causing variant will be affected, due to variable expressivity. This proposal will study both phenomena for the autosomal dominant disorder Alagille syndrome, caused by mutations in one of two genes in the Notch signaling pathway, the ligand JAGGED1 (JAG1) or the receptor, NOTCH2. Alagille syndrome is characterized by pediatric liver, heart, vertebral, renal, ocular, and facial anomalies with highly variable expressivity. The mechanism of disease for JAG1-related Alagille syndrome is haploinsufficiency whereas the mechanism for NOTCH2-related Alagille syndrome is less clear, with fewer reported variants, less functional characterization, and a higher prevalence of missense variants (>50%). Missense variants are difficult to classify, often requiring functional validation to support or reject pathogenicity. In Alagille syndrome, functional characterization has been carried out for only 19/125 reported missense mutations, thus, despite a high detection rate, the diagnostic rate is lower due to this uncertainty. We propose to resolve uncertainty in the diagnostics of Alagille syndrome using assays designed to characterize the pathogenicity of JAG1 and NOTCH2 missense variants and analysis of gene expression data from patient liver samples to identify gene expression signatures that can be used for genotype-phenotype evaluations. In Aim 1, we will design a Site Saturation Variant Library of all possible nucleotide permutations at each nucleotide position across a region with high missense variant uncertainty in the JAG1 C-terminus and test this library by developing a Multiplexed Assay for Variant Effects (MAVEs) that will measure cellular localization of JAG1 as a readout of protein function. In Aim 2, we will use FFPE liver tissue samples to analyze gene expression differences between Alagille syndrome patients and controls, as well as between Alagille syndrome patients with mild versus severe liver disease. In Aim 3, we will study the molecular basis of NOTCH2 variants through functional, expression, and enzymatic assays using mutant cell lines. We hypothesize that these proposed assays will identify a high-throughput method to test missense pathogenicity (Aim 1), identify gene expression differences between Alagille syndrome patients and controls as well as gene expression signatures that are different between Alagille syndrome patients with mild versus severe liver disease (Aim 2), and determine the mechanism by which NOTCH2 variants cause Alagille syndrome through functional analysis (Aim 3). Ultimately, these data will improve variant analysis for Alagille syndrome, improve our understanding of the molecular basis of liver disease in Alagille syndrome, and establish a framework for scalable classification of missense variants, delivering diagnostic information that can directly help clinicians.

项目摘要/摘要 基因组诊断的不确定性为实现基因组医学的全部潜力带来了障碍。 不确定性在：1）我们无法确定某些DNA变体是致病的还是良性的，2）我们 由于可变，无法预测一个患有疾病变异的人的程度将受到影响 表现力。该建议将研究常染色体显性疾病阿拉吉综合症的两个现象， 由Notch信号通路中的两个基因之一，配体Jagged1（Jag1）或 受体Notch2。阿拉吉综合症的特征是小儿肝，心脏，椎骨，肾脏，眼和 面部异常具有高度可变的表达性。 JAG1相关的Alagille疾病机制 综合征是单倍症，而与Notch2相关的Alagille综合征的机制不太清楚， 报道的变体较少，功能表征较少，错义变体的患病率较高 （> 50％）。错义变体很难分类，通常需要功能验证来支持或拒绝 致病性。在阿拉吉尔综合征中，仅报道了19/125的功能表征 因此，尽管检测率很高，但由于这种不确定性，诊断率较低。 我们建议使用旨在的测定法解决阿拉吉尔综合征诊断的不确定性 表征JAG1和Notch2错义变体的致病性以及基因表达数据的分析 从患者肝样品中识别可用于基因型 - 表型的基因表达特征 评估。在AIM 1中，我们将设计一个位点饱和变体库，该库的所有可能的核苷酸排列 JAG1 C末端中的每个区域中的每个核苷酸位置， 通过开发一种将测量细胞的变体效应（MAVE）来测试该库 JAG1的定位作为蛋白质功能的读数。在AIM 2中，我们将使用FFPE肝组织样品 分析Alagille综合征患者和对照组之间的基因表达差异，以及 阿拉吉综合征患者患有轻度肝脏疾病与严重肝病。在AIM 3中，我们将研究 使用突变细胞系通过功能，表达和酶法测定的Notch2变体。我们 假设这些提出的测定将确定一种测试错义致病性的高通量方法 （AIM 1），确定Alagille综合征患者与对照组以及基因之间的基因表达差异 在轻度肝与严重肝脏的阿拉吉尔综合征患者之间不同的表达信号 疾病（AIM 2），并确定Notch2变体引起Alagille综合征的机制 功能分析（目标3）。最终，这些数据将改善Alagille综合征的变体分析， 提高我们对阿拉吉综合征肝病分子基础的理解，并建立 一个可扩展的错义变体分类的框架，提供可以提供的诊断信息 直接帮助临床医生。