Eighty-one Sphynx cats diagnosed with hypertrophic cardiomyopathy by an echocardiographic examination performed by a board-certified veterinary cardiologist were recruited for evaluation. Cats were considered to be affected if they had a left ventricular end diastolic wall thickness of at least 0.6 cm for the interventricular septum and/or left ventricular free wall [22]. Thirteen domestic short haired cats over the age of 10 years were selected to serve as controls after evaluation by a board-certified veterinary cardiologist and being cleared of cardiomyopathy based on an echocardiographic identification of a left ventricular end diastolic wall dimension of less than 0.4 cm for the interventricular and/or left ventricular free wall.
DNA samples were obtained from each affected Sphynx cat and control cat. DNA samples from 214 cats with no known history of cardiac disease from 14 breeds (Bengal, Birman, British short hair, Burmese, Domestic Short Hair, Domestic Long Hair, Himalayan, Maine Coon, Manx, Norwegian Forest Cat, Persian, Ragdoll, Scottish Fold, Siamese) maintained in an archive at the NCSU College of Veterinary Medicine were also available for analysis. Approximately 3 µg of DNA from 14 affected Sphynx cats and the 13 control cats was submitted for library preparation and whole genome sequencing (WGS) at the University of North Carolina Chapel Hill High-Throughout Sequencing Facility (https://www.med.unc.edu/genomics). All sequencing experiments were designed as 125 base paired-end reads and samples were run on either 1 or 2 lanes of an Illumina HiSeq 2500 high-throughput sequencing system.
Read alignment and variant calling from WGS data were performed using a standardized bioinformatics pipeline for all samples as follows. Raw sequencing reads were quality checked for potential sequencing issues and contaminants using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter sequences, primers, Ns, and reads with quality score below 28 were trimmed using Trimmomatic [23, 24]. Reads with a remaining length of fewer than 20 base pairs after trimming were discarded. Post-trimmed reads were mapped to the Felis catus (Felis_catus 6.2) reference genome using BWA-MEM [25, 26]. Duplicated reads were marked using SAMBLASTER, and variants were called with Platypus [27,28,29]. The detected variants were annotated with dbSNP ID when available in dbSNP for Felis catus release 127 and variant effects were predicted with SnpEff based on Ensembl gene model release 75.
Variants present in affected cats were selected and filtered against a database of variants from the 13 control cats using the Variant Explorer tool of the Maverix Analytic Platform (http://www.maverixbio.com/). Variants were then categorized by Variant Effect Predictor 87 and prioritized for further study by their functional impact (e.g., stop codon, frameshift, indel, etc.) as well as potential cardiac involvement based on presence in a gene previously associated with a form of cardiomyopathy or a gene known to encode for proteins expressed in the myocardium, particularly those thought to be associated with myocardial function or development (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/).
Variants were then evaluated for likely pathogenic implications using the Standards and Guidelines for the Interpretation of Sequence Variants [30]. First, variants observed in more than 5% of the control population alleles were excluded. Next, a sequence was considered pathogenic if it was likely to be a truncating variant including gain of a stop codon, a frameshift indel, an altered splice site, an altered start codon, and/or a single or multi-exon deletion. Missense variants were considered to be significant if they altered a well conserved amino acid and were judged to have a likely deleterious impact with at least 2 of 3 in-silico programs including Polyphen (http://genetics.bwh.harvard.edu/pph2/), Sift (http://sift.jcvi.org/) and Provean (http://provean.jcvi.org/index.php). Finally, the data was visually inspected for multiallelic variations including larger insertions and deletions using the GenomeBrowse 2.1.2 (http://goldenhelix.com/products/GenomeBrowse/index.html) visualization tool.
The most promising variants were selected for further evaluation by Sanger Sequencing of DNA from 68 affected Sphynx diagnosed as previously described and the 214 cats with no known history of cardiac disease (controls). Finally, the variants were tested for allelic association with hypertrophic cardiomyopathy using a Fisher’s exact test. A p value of < 0.05 was considered significant. Penetrance and relative risk were determined for each of the most promising variants that were evaluated by Sanger Sequencing.
To better determine the potential impact of the variant at the muscular level, immunohistochemistry was performed for Ki67, a known marker of nuclear proliferative activity, and vimentin, to determine if any nuclear proliferative activity was associated with fibroblast or myocyte cells. More specifically, myocardial samples were collected immediately at time of death from 4 affected Sphynx cats and 3 mature non-sphynx control cats euthanized for non-cardiac disease. Samples were snap frozen at − 80° and 10 um cryostat sections were placed on charged glass slides. After fixing with 10% neutral buffered formalin, heat-induced epitope retrieval was performed (Thermo, #TA-135-HBL), and endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Tissues were blocked in 10% normal goat serum for one hour, then incubated overnight at 4 °C with rabbit anti-Ki67 antibody (Vector Laboratories, #VP-RM04; 1:100), followed by incubation with biotinylated goat anti-rabbit secondary antibody (Jackson #111-065-144; 1:500) for 1 h at room temperature. Signal was amplified using a Vectastain Elite ABC-HRP kit (Vector Laboratories, #PK-6100), and visualized using DAB (Thermp, #TA-QHDX-125). Tissues were blocked again in 10% normal goat serum for one hour, then incubated overnight at 4 °C with mouse anti-Vimentin antibody (Sigma, #V5255; 1:100), followed by incubation with biotinylated goat anti-mouse secondary antibody (Jackson #115-065-166) for one hour at room temperature. Signal was amplified using a Vectastain Elite ABC-AP kit (Vector Laboratories, #AK-5000), and visualized using Immpact Vector Red (Vector Laboratories, #SK-5105). Finally, tissues were counterstained with Hematoxylin, dehydrated using a series of graded alcohols, cleared with xylene and coverslipped with DPX (Electron Micrscopy Sciences, #13512). Using light microscopy, the amount of proliferative activity was evaluated blindly by one investigator (KM) by photographing each slide 5 times, randomizing the images and counting the number of Ki67 stained nuclei in 5 predefined areas (4 borders, center) of each slide at 4 × magnification. The number of stained nuclei was summed for each slide. The number of stained nuclei was compared between control cats and hypertrophic cardiomyopathy cats using the Student’s t test, with p < 0.05 considered to be significant.