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

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.