MYXOMATOUS MITRAL VALVE DISEASE IN CAVALIER KING CHARLES SPANIEL: CLINICAL, GENETIC, AND CARDIAC BIOMARKERS STUDY

One hundred and sixty-five CKCS subjects, both healthy and affected by different stages of MMVD, were examined from November 2018 to June 2021 at the Veterinary Teaching Hospital in Lodi – University of Milan – Cardiology Section. Each dog was submitted to a clinical examination and echocardiographic, radiographic, and morphometric data were collected. Whole blood and plasma were collected for genetic analysis and biomarker (miRNA) evaluation. The objectives of this study were: 1) To describe breed-specific reference values for vertebral heart score (VHS), vertebral heart atrial size (VLAS), M-VLAS, and radiographic left atrial enlargement (RLAD) in healthy adults CKCS; 2) To conduct a genomic study on a population of Italian CKCS; 3) To characterize echocardiographic features of the mitral valve in this breed, focusing on dogs classified as American College of Veterinary Internal Medicine (ACVIM) B1, without clinical signs and without left atrial and ventricular enlargement; 4) To analyze the relationships and the prognostic value of morphometric variables in CKCS affected by MMVD; 5) To analyze the expression of miRNAs described in the literature as being involved in the pathophysiology of MMVD, and identified in dogs’ plasma. Results: 1) Healthy CKCS had a median VHS of 10.08 ± 0.56v, a VLAS, M-VLAS and RLAD respectively of 1.79 ± 0.3v, 2.23 ± 0.44v and 1.2 ± 0.34v; 2). The top 1% single-nucleotide polymorphisms (SNPs) of both Wright’s fixation index (FST) and cross-population extended haplotype homozygosity (XP-EHH) analyses localized 10 consensus genes on chromosomes 3-11-14-19; 3) Within class B1, older subjects showed significantly higher values of anterior mitral valve area (AMVA), width (AMVW), mitral valve annulus in diastole (MVAd) and systole (MVAs) and lower sphericity index (SI); 4) A more severe mitral regurgitant jet size and a thicker anterior mitral valve leaflet were observed in CKCS smaller than standard proposed by the Ente Nazionale della Cinofilia Italiana (ENCI) and with morphometric characteristics tending to brachycephalism; 5) miR-30b-5 was significantly higher in ACVIM B1 compared to healthy subjects (ACVIM class A) and the AUC was 0.79. According to the age of dogs, the expression of miR-30b-5p remained significantly higher in group B1<3y (2.3 folds p=0.03), B1 3-7y (2.2 folds p=0.03), and B1>7y (2.7 folds p=0.02) than in stage A. The AUCs were fair in discriminating group B1<3y and A (AUC 0.78), and B1 3-7y and A (AUC 0.78), and good in discriminating group B1>7y and A (AUC 0.82). Conclusions: 1) Findings supported previous studies recommending the use of breed-specific reference values for VHS, VLAS, M-VLAS and RLAD and provided background data for future radiographic evaluations of CKCS dogs with clinical signs of cardiac disease; 2) This genetic analysis expands the knowledge of the genetic basis of MMVD by identifying genes involved in the early onset of MMVD in CKCS; 3) This is the first study that describes measurements of the anterior mitral valve leaflet and the mitral valve annulus in the CKCS affected by MMVD at different stages. Differently aged B1 dogs have different clinical and echocardiographic patterns. Further investigations with a larger study population and an appropriate follow-up would highlight prognostic factors related to disease worsening within this heterogeneous ACVIM class; 4) The morphological study of CKCS showed that a more severe regurgitant jet size was observed in subjects with a shorter head and nose. Subjects with a smaller head stop angle had thicker anterior mitral valve leaflets. Dogs with cephalic morphology more similar to the King Charles spaniel breed, that is with a brachycephalic morphotype, showed a more severe regurgitant jet size and valvular characteristics related to worse forms of MMVD (thicker anterior mitral valve leaflet, greater mitral valve annulus and lower sphericity index); 5) miR-30b-5p increases in the plasma of asymptomatic CKCS and this can be considered a potentially promising biomarker even at an asymptomatic stage of disease, particularly at a young age.