Developing a multiplex diagnostic test for SNPs related to dry eye disease

开发与干眼病相关的SNPs的多重诊断测试 项目总结： 干眼病(DED)是最常见的眼科疾病之一，全世界五分之一的成年人患有干眼病， 占美国成年人总数的5%-15%。DED的特征是泪膜渗透压升高和炎症。 在眼表，这表现为患者的各种不适症状。通知的一个主要目标 特别感兴趣的不是-EY-21-007是验证与发病风险相关的诊断生物标志物 无症状个体的死亡。由于复杂的病因机制，DED的诊断具有挑战性 这种疾病的根本原因是临床症状和体征之间的相关性很差，而且缺乏黄金标准 用于诊断。DED诊断和临床评估是主观的，通常取决于患者报告 症状。然而，仅根据症状进行诊断是不可靠的，因为DED的症状是重叠的 与其他眼部疾病的症状相一致。因此，非常需要可靠的方法来识别个人 有罹患DED的风险或支持DED的诊断。遗传因素导致了这些症状和 有DED的迹象。具体而言，IL1B、IL6R、MUC1、TNF-a、TNFAIP3、 而VDR基因被认为是与DED相关的危险因素。因此，发展一种核酸 对这些SNPs的扩增测试应该有助于识别高危个体，并支持DED诊断。基因标签 科技公司已经发明了几种具有新的差错防止机制的qPCR探针系统，这提供了 极佳的特异性。我们的内部DNA检测开关(IDDS)探针系统包括一个氟标记的探针 以及一种几乎与探针互补的猝灭剂标记的反探针。在没有预定的情况下 靶、探针和反探针结合在一起，从而猝灭荧光并防止假阳性结果。 在这项研究中，我们将使用IDDS探针系统生成DED风险等位基因的诊断测试。以特定的目标 1，我们将建立一种冷冻干燥的8管双IDDS探针检测与干眼病相关的SNPs。钥匙 目标1的里程碑是(I)开发与DED相关联的SNPs的双IDDS探针分析，(Ii)转换分析 到冻干形式，以及(Iii)确认在冻干后的每一次检测的性能。在具体目标2中， 我们将评估多重干眼病测试的关键性能特征。目标2个里程碑是 以确定双IDDS探头的(I)线性度、(Ii)检测极限(LOD)、(Iii)精密度和(Iv)稳定性 用人类基因组DNA样本进行分析，对每个感兴趣的等位基因进行纯合。在第一阶段取得成功将 支持扩大第二阶段测试，这将通过一个合同研究组织进行。同相 二、扩大化验特性(分析反应性、交叉反应性、携带、交叉污染、化验 将使用临床标本进行切割)。项目的成功将促进第一个项目的发展 对与DED易感性相关的SNP等位基因的分子诊断测试将支持患者的诊断和 改善患者护理。

Developing a multiplex diagnostic test for SNPs related to dry eye disease Confidential PI: Shafer, David A., PhD PROJECT SUMMARY: Dry eye disease (DED) is one of the most common ophthalmic conditions, affecting 1 in 5 adults worldwide and 5–15% of all adults in the United States. DED is characterized by increased tear film osmolarity and inflammation on the ocular surface, which manifests in patients as various symptoms of discomfort. A major objective of Notice of Special Interest NOT-EY-21-007 is to validate diagnostic biomarkers associated with the risk of developing DED in asymptomatic individuals. DED diagnosis is challenging due to the complex etiological mechanisms underlying the disease, a poor correlation between clinical signs and symptoms, and the lack of a gold standard for diagnosis. DED diagnosis and clinical evaluation are subjective and normally depend on patient-reported symptoms. However, diagnosis based on symptoms alone is unreliable because the symptoms of DED overlap with those of other ocular conditions. Thus, a significant need exists for reliable methods to identify individuals at risk for developing DED or to support the diagnosis of DED. Genetic factors contribute to the symptoms and signs of DED. Specifically, single-nucleotide polymorphisms (SNPs) in the IL1B, IL6R, MUC1, TNF-a, TNFAIP3, and VDR genes have been implicated as risk factors associated with DED. Therefore, developing a nucleic acid amplification test for these SNPs should help identify at-risk individuals and support DED diagnosis. GeneTAG Technology has invented several qPCR probe systems with a novel error-prevention mechanism, which provides excellent specificity. Our internal DNA-Detection Switch (iDDS) probe system comprises a fluor-labeled probe and a quencher-labeled antiprobe that is nearly complementary to the probe. In the absence of the intended target, the probe and antiprobe bind together, which quenches fluorescence and prevents false-positive results. In this study, we will generate a diagnostic test for DED risk alleles using the iDDS probe system. In Specific Aim 1, we will develop a lyophilized 8-tube dual-iDDS probe assay for SNPs linked to dry eye disease. The key milestones of Aim 1 are to (i) develop dual-iDDS probe assays for SNPs linked to DED, (ii) convert the assays to lyophilized format, and (iii) confirm the performance of each assay following lyophilization. In Specific Aim 2, we will evaluate key performance characteristics of the multiplex dry eye disease test. The Aim 2 milestones are to determine the (i) linearity, (ii) limit of detection (LoD), (iii) precision, and (iv) stability of the dual-iDDS probe assays with human genomic DNA samples, homozygous for each allele of interest. Success in Phase I will support expanded Phase II testing, which will be conducted through a contract research organization. In Phase II, expanded assay characterization (analytical reactivity, cross-reactivity, carryover, cross-contamination, assay cut-off) will be performed using clinical specimens. Project success will facilitate development of the first molecular diagnostic test for SNP alleles linked to DED susceptibility, which will support patient diagnosis and improve patient care.