Multi-omic analyses in Abyssinian cats with primary renal amyloid deposits

Abstract

The amyloidoses constitute a group of diseases occurring in humans and animals that are characterized by abnormal deposits of aggregated proteins in organs, affecting their structure and function. In the Abyssinian cat breed, a familial form of renal amyloidosis has been described. In this study, multi-omics analyses were applied and integrated to explore some aspects of the unknown pathogenetic processes in cats. Whole-genome sequences of two affected Abyssinians and 195 controls of other breeds (part of the 99 Lives initiative) were screened to prioritize potential disease-associated variants. Proteome and miRNAome from formalin-fixed paraffin-embedded kidney specimens of fully necropsied Abyssinian cats, three affected and three non-amyloidosis-affected were characterized. While the trigger of the disorder remains unclear, overall, (i) 35,960 genomic variants were detected; (ii) 215 and 56 proteins were identified as exclusive or overexpressed in the affected and control kidneys, respectively; (iii) 60 miRNAs were differentially expressed, 20 of which are newly described. With omics data integration, the general conclusions are: (i) the familial amyloid renal form in Abyssinians is not a simple monogenic trait; (ii) amyloid deposition is not triggered by mutated amyloidogenic proteins but is a mix of proteins codified by wild-type genes; (iii) the form is biochemically classifiable as AA amyloidosis.

Figure 1

Overview of the different types of variants identified by Whole Genome Sequencing in the two affected Abyssinian cats compared to the Control Population with the Golden helix SVS Software. Variants distribution is reported for each cat chromosome. Variants referred to the class of “Other” included 5′UTR variants, downstream and upstream gene variants, non-coding transcript exon variants, and splice region variants.

Figure 2

Venn diagram of the proteins identified in FFPE kidney tissues from cats affected by renal amyloidosis (AMY) and healthy (CTR). Only proteins present and quantified in at least 2 out of 3 repeats in each group were considered as positively identified.

Figure 3

Workflow of the proteomic approach. A shotgun proteomic analysis was performed on FFPE kidney tissue from cats affected by renal amyloidosis (Affected) and healthy (Control). Statistical analyses were performed using the Perseus software (version 1.4.0.8, www.biochem.mpg.de/mann/tools/).

Figure 4

MDS plot realized with R Studio (https://www.rstudio.com/products/rpackages/), showing the cluster of the affected cats, based on the 258 miRNAs expression (log2 counts per million). Affected individuals (purple) are grouped on the left, while the healthy individuals are represented by grey dots on the right.

Figure 5

Heatmap created using the R package "gplots", version 3.03 (https://rstudio.com/products/rpackages/) with the 60 significant miRNAs (nominal P-val < 0.05). The grey bar and the purple bar represent the healthy (A4, A5, and A6) and the affected (A1, A2, A3) Abyssinians, respectively. The shades of blue refer to down-regulated miRNAs while the shades of red refer to up-regulated miRNAs.

Figure 6

Network plot showing the interactions in pathological kidney tissue among the proteins exclusively present or up-regulated in the affected specimens and the miRNAs identified in the study, according to miRNet database. MiRNAs circled in red represent the only four miRNAs regulating exclusively the proteins related to the affected cats and none of those found in the controls.