Vladimir Tsivinsky

Analysis of organ-enriched microRNAs in plasma as an approach to development of Universal Screening Test: feasibility study

BackgroundEarly disease detection with a minimally invasive screening test will significantly increase effectiveness and decrease the cost of treatment. Here we propose a framework of a novel approach – Universal Screening Test (UST) for the detection of pathological processes in a particular organ system, organ, or tissue by RT-qPCR analysis of circulating cell-free miRNAs in plasma. As the first step towards assessing the feasibility of this concept, the present study was designed to analyze whether the same microRNAs (miRNAs) can detect various diseases of a particular organ system.MethodsRNA was extracted from plasma using Trizol treatment and silica binding. Levels of miRNAs were measured by single target RT-qPCR. The following innovations have been tested and proven effective: (i) the use of organ system/organ/tissue-enriched miRNAs; (ii) the use of miRNAs associated with broad disease categories, such as cancer and inflammation, in combination with the organ-enriched miRNAs; and (iii) the use of “miRNA pairs” for selecting miRNA combinations with the highest sensitivity and specificity.ResultsHere we report biomarker miRNA pairs effectively differentiating (i) patients with pulmonary system diseases (asthma, pneumonia and non-small cell lung cancer) and gastrointestinal (GI) system diseases (Crohn’s disease, stages I/II esophageal, gastric and colon cancers) from controls, each with 95% accuracy; (ii) patients with a pathology of the pulmonary system from patients with a pathology of the GI system with 94% accuracy; and (iii) cancer patients (stages I/II esophageal, gastric, colon cancers, or non-small cell lung cancer) from patients with inflammatory diseases (asthma, pneumonia, or Crohn’s disease) with 93%-95% accuracy.ConclusionsThe results obtained in the present study, along with the data reported by us and others previously, are encouraging and lay the ground for further investigation of the described approach for UST development.

Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative diseases

BackgroundMinimally invasive specific biomarkers of neurodegenerative diseases (NDs) would facilitate patient selection and disease progression monitoring. We describe the assessment of circulating brain-enriched microRNAs as potential biomarkers for Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS).MethodsIn this case-control study, the plasma samples were collected from 250 research participants with a clinical diagnosis of AD, FTD, PD, and ALS, as well as from age- and sex-matched control subjects (n = 50 for each group), recruited from 2003 to 2015 at the University of Pennsylvania Health System, including the Alzheimer’s Disease Center, the Parkinson’s Disease and Movement Disorders Center, the Frontotemporal Degeneration Center, and the Amyotrophic Lateral Sclerosis Clinic. Each group was randomly divided into training and confirmation sets of equal size. To evaluate the potential of circulating microRNAs enriched in specific brain regions affected by NDs and present in synapses as biomarkers of NDs, the levels of 37 brain-enriched and inflammation-associated microRNAs in the plasma of all participants were measured using individual qRT-PCR. A “microRNA pair” approach was used for data normalization.ResultsMicroRNA pairs and their combinations (classifiers) capable of differentiating NDs from control and from each other were defined using independently and jointly analyzed training and confirmation datasets. AD, PD, FTD, and ALS are differentiated from control with accuracy of 0.89, 0.90, 0.88, and 0.83 (AUCs, 0.96, 0.96, 0.94, and 0.93), respectively; NDs are differentiated from each other with accuracy ranging from 0.77 (AUC, 0.87) for AD vs. FTD to 0.93 (AUC, 0.98) for AD vs. ALS. The data further indicate sex dependence of some microRNA markers. The average increase in accuracy in distinguishing ND from control for all and male/female groups is 0.06; the largest increase is for ALS, from 0.83 for all participants to 0.92/0.98 for male/female participants.ConclusionsThe work presented here suggests the possibility of developing microRNA-based diagnostics for detection and differentiation of NDs. Larger multicenter clinical studies are needed to further evaluate circulating brain-enriched microRNAs as biomarkers for NDs and to investigate their association with other ND biomarkers in clinical trial settings.

DETECTION AND DIFFERENTIATION OF MILD COGNITIVE IMPAIRMENT, ALZHEIMER'S AND PARKINSON’S DISEASES BY ANALYSIS OF BRAIN-ENRICHED MICRORNAS IN PLASMA

Background: Recent genome-wide association studies (GWAS) have identified around 20 variants as late-onset Alzheimer’s disease (LOAD) susceptibility loci in whites. In addition to these single loci tests, it is important to detect and understand combined effects of multiple associated genes on LOAD. We performed a preliminary network analysis incorporating human protein-protein interaction database mined from 12 different sites including BIND, BioGRID, IntAct etc. to the HapMap2-imputed combined ADGC data set. Post-GWAS, this helps researchers to prioritize functionally related genes and networks that are of the highest biological relevance underlying the pathogenesis of LOAD. Methods: We combined HapMap2-imputed data sets from 15 studies after performing strict quality control. We performed a case-control association for LOAD adjusting for population sub-structure and study sites on a set of 19,692 unrelated individuals using PLINK and those results were used to perform a gene-wide analysis using VEGAS. The gene-wide association results were then integrated into the human protein-protein interaction network using a dense module searching (DMS) method to identify candidate genes or sub-networks for LOAD. We then attempted to functionally validate candidate genes from this network in vivo using a transgenic C. elegans model of Ab 1-42 toxicity. Results: The network analysis identified several of the known LOAD risk loci as well as other genes such as ALB, BAG1 and UBC to be strongly associated with LOAD. RNAi knockdown of the C. elegans orthologs of UBC (ubq-1 or ubq-2) significantly accelerated the age-associated onset of Ab 1-42 toxicity. Conclusions:We were able to identify a set of significant modules and candidate genes, including some well-studied genes not detected in the single-marker analysis of GWA studies for LOAD, and to demonstrate a role for two of these genes as modifiers of Ab toxicity in C. elegans. This approach provides complementary data to a GWAS of a complex disease phenotype by incorporating biological knowledge derived from protein-protein interactions and allows for initial functional validation in vivo. Further functional enrichment analysis is needed to determine whether these novel loci may provide targets for interventions to ameliorate LOAD. THURSDAY, JULY 17, 2014 ORAL SESSIONS O5-05 BIOMARKERS: NOVEL MOLECULAR FLUID MARKERS

Early detection of Alzheimer's disease by analysis of brain-enriched miRNA biomarkers in plasma

IL7was significantly negatively associated with depression (OR1⁄40.01, 95% CI .00-0.47). Serum IL7 was significantly positively associated with depression (OR1⁄43.01, 95% CI 1.02-9.31). Preliminary analyses suggest that diagnosis (AD versus control) also impacted the relation between GDS scores and IL7. Plasma IL7 levels were significantly negatively associated with GDS scores among women diagnosed with AD whereas serum IL7 was significantly negatively associated with GDS scores among female normal controls. Conclusions: Our results confirm prior work that IL7 is significantly associated with depression scores and shows that the IL7 depression link varies by gender and blood fraction. IL7 levels are weakly correlated across blood fractions. Future work examining blood-based markers of depression must consider gender as well as blood fraction in analyses. If IL-7 is considered in as a therapeutic agent for depression or AD, the current findings suggest that gender should be considered in trial design.