Abhijit Sreepada ¹ Rasul Khasanov ² Enas Zoheer Elkrewi ^1,2 Carolina De La Torre ³ Judith Felcht ² Ahmad A. Al Abdulqader ^2,4 Richard Martel ² Nicolás Andrés Hoyos ² Michael Boettcher ² Lucas Wessel ² Karl-Herbert Schäfer ^5* Maria Angeles Tapia-Laliena ^2*

• ¹ Translational Medical Research/International Master in Innovative Medicine” Master Program, Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Germany, Mannheim, Baden-Württemberg, Germany
• ² Department of Pediatric Surgery, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Baden-Württemberg, Germany
• ³ NGS Core Facility, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany, Mannheim, Germany
• ⁴ Department of Surgery, College of Medicine, King Faisal University, Al Hofuf, Saudi Arabia, Al Hofuf, Saudi Arabia
• ⁵ Working group Enteric Nervous Systems (AGENS), University of Applied Sciences Kaiserslautern, Campus Zweibrücken, Zweibrücken, Germany

Hirschsprung’s disease (HSCR) is characterized by congenital absence of ganglion cells in the gastrointestinal tract, which leads to impaired defecation, constipation and intestinal obstruction. HSCR current diagnosis is based on Rectal Suction Biopsies (RSBs), which could be complex in newborns. Occasionally, there is a delay in diagnosis that can increase the risk of clinical complications. Consequently, there is room for new non-invasive diagnostic methods that are objective, more logistically feasible and also delivering a far earlier base for a potential surgical intervention. In recent years, microRNA (miRNA) came into the focus as a relevant early marker that could provide more insights into the etiology and progression of diseases. Therefore, in the search of a non-invasive HSCR biomarker, we analyzed miRNA expression in urine samples of HSCR patients. Results from 5 HSCR patients using microarrays, revealed hsa-miR-378h, hsa-miR-210-5p, hsa-miR-6876-3p, hsa-miR-634 and hsa-miR-6883-3p as the most upregulated miRNAs; while hsa-miR-4443, hsa-miR-22-3p, hsa-miR-4732-5p, hsa-miR-3187-5p, hsa-miR-371b-5p where the most downregulated miRNAs. Further search in miRNAwalk and miRDB databases showed that certainly most of these dysregulated miRNAs identified target HSCR associated genes, such us RET, GDNF, BDNF, EDN3, EDNRB, ERBB, NRG1, SOX10; and other genes implied in neuronal migration and neurogenesis. Finally, we could also validate some of these miRNA changes in HSCR urine by RT-qPCR. Altogether, our analyzed HSCR cohort present a dysregulated miRNA expression pattern that can be detected in urine. Our findings open the possibility of using specific urine miRNA signatures as non-invasive HSCR diagnosis method in the future.