Tear Fluid as a Biomarker for Parkinson’s Disease: Downregulation of DNA Repair Genes/Pathways via RNA-Seq Analysis
Mark Lew1, Audrey Omidsalar3, Shruti Singh Kakan4, Daniel Gerke5, Maryam Tanveer3, Danielle Feigenbaum2, Emily Tamadonfar3, Sarah Hamm-Alvarez3, Brooke Hjelm3
1Neurology, Keck/USC School of Medicine, 2Keck/USC School of Medicine, 3Keck /USC School of Medicine, 4USC School of Pharmacy, 5USC/Harlyne J Norris Cancer Research Tower
Objective:

We performed an RNA-Seq analysis of tears from 16 Parkinson’s Disease (PD) patients and 16 healthy, age-matched healthy controls (HC) to identify genes and pathways that may serve as biomarkers of PD.

Background:

We previously demonstrated that PD tears have significantly higher levels of oligomeric a-synuclein than HC tears. Analyses of hundreds of subjects demonstrated this biomarker predicted PD diagnosis, irrespective of disease duration (AUROC=0.76). To improve this model, we performed RNA-Seq on PD and HC tears. Pilot studies of brain and tears revealed ~74% of genes expressed in aged cortex were expressed in tears.

 

Design/Methods:

Schirmer’s Tear Strips were frozen at -80°C. RNA was extracted and evaluated for quantity and integrity (RIN). RNA-Seq libraries were prepared with the NEBNext Ultra II RNA Library Prep Kit and sequenced on an Illumina NovaSeq sequencer. FASTQ files were aligned with Tophat and transcripts were counted and compared with Cufflinks. Pathway analysis was performed using the Qiagen Ingenuity Pathway Analysis (IPA) software and unsupervised, hierarchical clustering of significantly affected pathways was tested with RStudio.

Results:

RNA-Seq analysis showed more genes were downregulated in PD tears (N=898) vs. upregulated (N=238). IPA analysis of downregulated genes revealed DNA repair pathways were significantly affected, and reflected the downregulation of 60 genes related to DNA excision repair, recombination, and damage in PD tears. These 60 genes included HNRNPC, TARDBP, USP47, EZH2, FANCC. Unsupervised hierarchical clustering of these 60 genes’ transcript levels (FPKMs) separated PD and HC with 83.9% accuracy (sensitivity 93.3% and specificity 75%).

Conclusions:
RNA-Seq may provide a useful strategy for PD diagnosis using tears, a non-invasive biofluid. Preliminary data suggests increased diagnostic accuracy compared to our tear investigations of oligomeric a-synuclein levels alone. Downregulation of DNA repair genes/pathways suggests that alterations observed in PD brains and dopaminergic neurons are also observed in tears
10.1212/WNL.0000000000202879