A chromosome-scale fishing cat reference genome for the evaluation of potential germline risk variants

Abstract

The fishing cat, Prionailurus viverrinus, faces a population decline, increasing the importance of maintaining healthy zoo populations. Unfortunately, zoo-managed individuals currently face a high prevalence of transitional cell carcinoma (TCC), a form of bladder cancer. To investigate the genetics of inherited diseases among captive fishing cats, we present a chromosome-scale assembly, generate the pedigree of the zoo-managed population, reaffirm the close genetic relationship with the Asian leopard cat (Prionailurus bengalensis), and identify 7.4 million single nucleotide variants (SNVs) and 23,432 structural variants (SVs) from whole genome sequencing (WGS) data of healthy and TCC cats. Only BRCA2 was found to have a high recurrent number of missense mutations in fishing cats diagnosed with TCC when compared to inherited human cancer risk variants. These new fishing cat genomic resources will aid conservation efforts to improve their genetic fitness and enhance the comparative study of feline genomes.

Figure 1

The pedigree and geographic origins of founder individuals. (a) North American fishing cat population pedigree. In total there are 161 cats in the historic population, with 25 of those being cats currently in zoo-managed care and 15 total cats being confirmed as TCC cases. (b) Geographical origins of the current North American zoo fishing cat population individuals according to studbook information. Of the 16 founder cats, 6 were from Sri Lanka, 5 from Thailand, and 2 from Cambodia. The remaining 3 were documented as being from Asia, however, no specific country of origin is indicated. (c) Pictured here is the fishing cat selected for the reference genome assembly. Anna (AZA Studbook #950). Initially born in the La Fleche Zoo on 9/14/2010. After being transferred to two other European facilities in Bucharest, Romania, she eventually joined the AZA population in 2010 at the Chicago Zoological Society. She was then transferred to the Oklahoma City Zoo in 2020. Photograph provided by Animal Care Specialist Natalie Farley, Chicago Zoological Society. Figure adapted from “BioRender Templates—Create New”, by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates.

Figure 2

Chromosome alignments between the Asian leopard cat and fishing cat. (a) Synteny plot comparing the Asian leopard cat chromosomes (ALC) to the fishing cat chromosomes (FC). Most alignments pictured are found to exhibit a high level of synteny with each other. (b) Examining alignments between the Asian leopard cat and fishing cat, the chromosome with an interesting alignment is in D1 as indicated by this SafFire output. D1 alignments illustrate an inversion ~ 6.0 Mb in size towards the end of the alignment.

Figure 3

Missense variants identified in the fishing cat cohort. (a) A fishing cat urinary tract. Indicated by the black circle is a tumor excised from the bladder wall of a transitional cell carcinoma affected fishing cat. Tumor photograph provided by Dr. Leslie Lyons, University of Missouri. (b) Depicted in this bar graph are the variant types identified in the fishing cat cohort. The most prevalent were found to be intronic and intergenic variants, representing 65.31% and 21.28% respectively. Additionally, downstream variants and upstream variants were also present at 5.48% and 5.51%. Lastly, additional variants such as exon, gene, splice site acceptor, splice site donor, transcript, UTR 3 prim, and UTR 5 prime collectively comprised of 2.43% of all variants identified. (c) This lollipop plot illustrates six of the missense locations present in all cancer cats in BRCA2 (LOC125162678) and the single missense positions in DICER1 (LOC125167827), COL7A1 (LOC125159913), and FAH (LOC125158967). All small red lollipops on all genes indicate positions more strongly correlated with cancer cats. For BRCA2 all cancer cats expressed missense mutations at all indicated locations, with some normal-presenting cats also experiencing missense variants. D2392G on BRCA2 is indicated by a larger green lollipop as the heterozygous genotype is unique to only cancer cats. At the five other BRCA2 positions 40% of all control cats are also affected. Position G1276 on DICER1 shows 83% of cancer and 20% of control affected. Position H50 on COL7A1 shows 100% cancer 60% control cats affected. Position R217 on FAH shows 83% cancer and 20% of control cats affected. All sites on all genes experienced an amino acid change because of the base pair changes. Figure adapted from “BioRender Templates-Create New”, by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates.

Figure 4

Structural variant analysis of coding regions. (a) This stacked bar plot depicts the classification of SV types (deletions, insertions, and duplications in the following genomic regions: upstream, downstream, splice site, 3’ UTR, 5’ UTR, exon, intergenic, and intronic regions. Of the total 23,432 SVs, 22,419 were classified as deletions, 910 duplications, and 103 inversions. Of the variant classifications, intergenic and intronic were the most prominent with 10,638 and 9498 being identified respectively. (b) This tumorigenesis pathway includes four genes (highlighted in green) containing coding region SVs present in the cohort. At least one cancer cat had to contain the SV to be examined in this analysis. Of the four genes, two that were only present in cancer cats were MCIDAS and ARAP1.