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 基因组诊断中的不确定性为实现基因组医学的全部潜力制造了障碍。 不确定性明显体现在：1)我们无法确定某些DNA变异是致病的还是良性的；2)我们的 由于变量的原因，无法预测携带致病变异的人会受到多大程度的影响 表现力。这项建议将研究常染色体显性遗传病Alagille综合征的两种现象， 由Notch信号通路中两个基因中的一个突变引起的，即配体Jagged1(JAG1)或 受体，NOTCH2。Alagille综合征的特征是儿科的肝、心、脊椎、肾、眼和 面部异常具有高度可变的表现力。JAG1相关Alagille的致病机制 综合征是单倍体功能不全，而NOTCH2相关的阿拉格尔综合征的机制不太清楚， 报告的变异较少，功能特征较少，错义变异的流行率较高 (&gt；50%)。错义变体很难分类，通常需要功能验证才能支持或拒绝 致病性。在Alagille综合征中，只有19/125的报告进行了功能表征 错义突变，因此，尽管检测率很高，但由于这种不确定性，诊断率较低。 我们建议使用旨在解决Alagille综合征诊断的不确定性的分析方法 JAG1和NOTCH2错义变异体的致病性鉴定及基因表达数据分析 从患者肝脏样本中识别可用于基因表型的基因表达特征 评估。在目标1中，我们将设计一个包含所有可能的核苷酸排列的位点饱和变异库 JAG1 C-末端具有高度错义变异不确定性的区域的每个核苷酸位置和 通过开发一种用于测量细胞变异效应的多路测试(MAVE)来测试这个库 将JAG1定位为蛋白质功能的读数。在目标2中，我们将使用FFPE肝组织样本 分析Alagille综合征患者和对照组之间以及两者之间的基因表达差异 Alagille综合征患者合并轻度肝病和重度肝病。在目标3中，我们将研究分子基础 Notch2变异体通过使用突变细胞系进行的功能、表达和酶分析。我们 假设这些建议的化验将确定一种高通量方法来测试错义致病性 (目的1)确定Alagille综合征患者和对照组之间的基因表达差异以及基因 Alagille综合征轻度和重度肝病患者的表达特征不同 疾病(目标2)，并确定NOTCH2变异通过以下途径导致Alagille综合征的机制 功能分析(目标3)。最终，这些数据将改进阿拉吉尔综合征的变异分析， 提高我们对Alagille综合征肝脏疾病的分子基础的理解，并建立 可扩展的错义变体分类框架，可提供以下诊断信息 直接帮助临床医生。