Identification of miRNAs and evaluation of their differential expression in Abyssinian cat amyloidosis

Introduction: Domestic felids represent one of the main species in which amyloidosis occurs. The disease is caused by the presence of protein complexes, known as amyloids, which form insoluble deposits in different organs. However, little is known about the pathogenic pathway and the genetic of the disease is still under exploration. Among cat breeds, amyloidosis particularly affects Abyssinian/Somali and Siamese/Oriental cats, where the main target organs for the deposit are kidneys and liver, respectively. Objective: The aim of this study is i) to detect miRNAs expressed in amyloidosis affected and healthy Abyssinian kidneys ii) to evaluate their possible differential expression iii) to identify miRNAs potentially involved in the disease onset or in the regulation of its pathogenesis. Materials & Methods: miRNAs were extracted from Formalin Fixed Paraffin Embedded (FFPE) kidney slides collected from 6 affected and 4 healthy Abyssinians using the miRNeasy Mini Kit (Qiagen). The sequencing of miRNAs was carried out by smallRNA-seq kit (Illumina) and Illumina NextSeq500 platform, and its quality was assessed with FastQC. Cutadapter was used to remove the adapter sequences from the high-throughput sequencing reads. MiRDeep2 was then used to map reads against the cat reference genome (Felis catus, genome assembly version 9.0), identify putative miRNAs, quantify their expression and identify homologous human miRNAs. MiRNAs with less than 10 reads for each sample were filtered out. Raw counts were normalized with TMM method (Trimmed mean of M values) and expressed as log2 CPM (counts per million). Differential expression was assessed with a moderated t test (limma) and nominal p values were adjusted by Benjamini-Hochberg method. Results: A total of 854 miRNAs were detected, and 341 miRNAs were selected as representative after filtering. A total of 22 miRNAs showed significant expression difference between affected and healthy Abyssinians (p<0.05), but none of this was significant after p-value correction for multiple hypotheses. A total of 6 (out of 22) are known to be involved in the development of Alzheimer Disease (AD), four of which with a P-value < 0.009. Suggestively, within the not significantly associated to amyloidosis miRNAs (p>0.05), miR- 26a-5p (P-value 0.120) is one of the main miRNAs involved in the human immunoglobulin light chain (AL) amyloidosis onset. Conclusions: recent studies in humans have been focusing on disclosing the potential role of miRNAs in the accumulation of amyloid fibrils, especially in AD. It was shown that miRNAs dysregulation plays an important role in the disease alterations, although it cannot be considered the main trigger of the AD itself. Some of the identified miRNAs in the present study were already known to be associated with human AD. These results are encouraging and will help understanding the pathogenesis of feline amyloidosis. The genes directly regulated and involved in these pathways are under investigation and further evidences will be obtained with an integrative approach through a proteomic analysis.