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

开发与干眼病相关的SNPs的多重诊断测试机密PI：Shafer，大卫A.，博士 项目概要： 干眼病（DED）是最常见的眼科病症之一，影响全世界五分之一的成年人， 占美国成年人的5-15%。DED的特征是泪膜渗透压增加和炎症 在眼表面上，其在患者中表现为各种不适症状。《通知》的一个主要目标 特别关注的NOT-EY-21-007是验证与发生以下疾病风险相关的诊断生物标志物： 无症状个体中的DED。DED的诊断是具有挑战性的，由于复杂的病因机制 潜在的疾病，临床体征和症状之间的相关性差，以及缺乏金标准 进行诊断。DED诊断和临床评价是主观的，通常取决于患者报告的 症状然而，仅基于症状的诊断是不可靠的，因为DED的症状重叠 与其他眼部疾病的患者相同。因此，非常需要可靠的方法来识别个体 有发展DED的风险或支持DED的诊断。遗传因素导致症状， 的迹象。具体而言，IL 1B、IL 6 R、MUC 1、TNF-α、TNFAIP 3、 和VDR基因被认为是与DED相关的危险因素。因此，开发核酸 这些SNPs的扩增测试应该有助于识别风险个体并支持DED诊断。基因标签 技术已经发明了几种具有新型防错机制的qPCR探针系统，其提供了 特异性极佳。我们的内部DNA检测开关（iDDS）探针系统包括荧光标记探针 和与探针几乎互补的淬灭剂标记的反探针。在没有预期的 当检测到靶点时，探针和反探针结合在一起，从而淬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。具体目标 1，我们将开发一种冻干的8管双iDDS探针检测与干眼病相关的SNP。关键 目标1的里程碑是（i）开发与DED相关的SNP的双iDDS探针测定法，（ii）将测定法 至冻干形式，和（iii）确认冻干后各测定的性能。在具体目标2中， 我们将评估多重干眼症测试的关键性能特征。Aim 2的里程碑是 确定双iDDS探针的（i）线性、（ii）检测限（LoD）、（iii）精密度和（iv）稳定性 使用人类基因组DNA样品进行的分析，对于每个感兴趣的等位基因是纯合的。第一阶段的成功将 支持扩大的第二阶段测试，这将通过合同研究组织进行。同相 II，扩展的检测试剂盒表征（分析反应性、交叉反应性、残留、交叉污染、检测 临界值）将使用临床样本进行。项目的成功将促进第一个 与DED易感性相关的SNP等位基因的分子诊断测试，这将支持患者诊断， 改善病人护理。

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.