Transcriptomic profiling of Cytomegalovirus Infection in Cardiac Transplantation: proof-of-concept for a new strategy in tissue markers application

Ilaria Barison¹Diego Perazzolo¹Chiara Castellani¹Alessia Giarraputo¹Elisabetta Rossi^2,3Luca Vedovelli⁴Sonia Anna Minuzzo²Chiara Tessari⁵Nicola Pradegan⁵Giuseppe Toscano⁵Francesco Tona⁶Cristina Basso¹Gino Gerosa⁵Susanna Mandruzzato^2,3Davide A. Abate, MD PhD⁷Dario Gregori⁴Annalisa Angelini^1*Marny Fedrigo¹

• ¹Cardiovascular Pathology, Department of Cardiac, Thoracic and Vascular Sciences and Public Health, University of Padova, Padua, Veneto, Italy
• ²Department of Surgery, Oncology and Gastroenterology, Oncology Section, University of Padova, Padua, Veneto, Italy
• ³Immunology and Molecular Oncology Diagnostics, Veneto Institute of Oncology IOV - Istituto di Ricovero e Cura a Carattere Scientifico, Padua, Veneto, Italy
• ⁴Unit of Biostatistics, Epidemiology and Public Health, Department of Cardiac, Thoracic, Vascular Sciences, and Public Health, University of Padova, Padua, Veneto, Italy
• ⁵Division of Cardiac Surgery, Department of Cardiac, Thoracic and Vascular Sciences and Public Health, University of Padova, Padua, Veneto, Italy
• ⁶Cardiology Unit, Department of Cardiac, Thoracic, Vascular Sciences and Public Health, University of Padua, Padua, Veneto, Italy
• ⁷Department of Molecular Medicine, University of Padova, Padua, Veneto, Italy

Background: Cytomegalovirus (CMV) infection is a relevant threat to heart-transplanted patients during the first year after surgery, leading to increased morbidity and, in some cases, mortality. This proof-of-concept study aims to assess the transcriptomic profile of CMV infection in cardiac transplanted patients as a new diagnostic approach to discriminate infection and Acute Cellular Rejection (ACR) on EMB specimens.Methods: We performed a microarray-based messenger RNA (mRNA) and micro-RNA (miRNA) profiling. We analyzed three patient groups in the setting of CMV viremia and inflammatory infiltrate: a control group (n=5), an ACR group (n=5), and an infection group (n=6). Differentially expressed mRNA and miRNA were further investigated through bioinformatic pathway analysis. Results: Focusing on infection vs rejection comparison, we investigated the role of the 18 differentially expressed mRNAs and the 12 miRNAs with the most significative p-value (gene level fold change, FC <-2 or >2, p-value <0.05). Based on the bioinformatic analysis, we explored the regulatory effects of these miRNAs on the mRNA pathways independently identified in the same samples. The results showed that two genes, IL7R and GZMK (-38.63 and -3.15 FC, respectively), and two miRNAs, mir-93-5p and mir-345-5p (-2.63 and -2.18 FC, respectively), are differentially expressed in infection and can be exploited to differentiate CMV-positive from ACR-positive EMB specimens, reaching an AUC of 0.87 and an accuracy of 91% at cross-validation.Conclusions: We have identified a distinctive combined molecular profile of mRNAs and miRNAs for infection in post-cardiac transplant follow-up. Based on IL7R, GZMK, mir-93-5p, and mir-345-5p we suggest a novel possible workflow to distinguish infection, where those markers are downregulated, from rejection, where they are overexpressed, on EMB specimens. This analysis showed good accuracy and promising predictive performance. The future combined analysis of these genes and these miRNAs through user-friendly techniques, such as quantitative PCR, could reduce turn-around time and improve our diagnostic power for distinguishing CMV infection from ACR in EMB specimens.