Classification of feline hypertrophic cardiomyopathy-associated gene variants according to the American College of Medical Genetics and Genomics guidelines.

Introduction: The correct labeling of a genetic variant as pathogenic is important as breeding decisions based on incorrect DNA tests can lead to the unwarranted exclusion of animals, potentially compromising the long-term health of a population. In human medicine, the American college of Medical Genetics (ACMG) guidelines provide a framework for variant classification. This study aims to apply these guidelines to six genetic variants associated with hypertrophic cardiomyopathy (HCM) in certain cat breeds and to propose a modified criterion for variant classification. Methods: Genetic samples were sourced from five cat breeds: Maine Coon, Sphynx, Ragdoll, Devon Rex, and British Short- and Longhair. Allele frequencies were determined, and in the subset with phenotypes available, odds ratios to determine the association with HCM were calculated. In silico evaluation followed with joint evidence and data from other publications assisting in the classification of each variant. Results: Two variants, MYBPC3:c.91G > C [A31P] and MYBPC3:c.2453C > T [R818W], were designated as pathogenic. One variant, MYH7:c.5647G > A [E1883K], was found likely pathogenic, while the remaining three were labeled as variants of unknown significance. Discussion: Routine genetic testing is advised solely for the MYBPC3:c.91G > C [A31P] in the Maine Coon and MYBPC3:c.2453C > T [R818W] in the Ragdoll breed. The human ACMG guidelines serve as a suitable foundational tool to ascertain which variants to include; however, refining them for application in veterinary medicine might be beneficial.

Identification of novel genetic risk factors of dilated cardiomyopathy: from canine to human.

BACKGROUND: Dilated cardiomyopathy (DCM) is a life-threatening heart disease and a common cause of heart failure due to systolic dysfunction and subsequent left or biventricular dilatation. A significant number of cases have a genetic etiology; however, as a complex disease, the exact genetic risk factors are largely unknown, and many patients remain without a molecular diagnosis. METHODS: We performed GWAS followed by whole-genome, transcriptome, and immunohistochemical analyses in a spontaneously occurring canine model of DCM. Canine gene discovery was followed up in three human DCM cohorts. RESULTS: Our results revealed two independent additive loci associated with the typical DCM phenotype comprising left ventricular systolic dysfunction and dilatation. We highlight two novel candidate genes, RNF207 and PRKAA2, known for their involvement in cardiac action potentials, energy homeostasis, and morphology. We further illustrate the distinct genetic etiologies underlying the typical DCM phenotype and ventricular premature contractions. Finally, we followed up on the canine discoveries in human DCM patients and discovered candidate variants in our two novel genes. CONCLUSIONS: Collectively, our study yields insight into the molecular pathophysiology of DCM and provides a large animal model for preclinical studies.

Evaluation of cardiac troponin I as a predictor of death in critically ill cats.

BACKGROUND: Abnormally high serum cardiac troponin I (cTnI) concentration, reflecting leakage from or necrosis of cardiomyocytes, is a negative prognosticator for death in dogs. OBJECTIVES: To investigate in critically ill cats whether serum cTnI concentration is abnormally high, identify conditions associated with abnormally high cTnI concentrations, and evaluate cTnI as an independent prognosticator for death and a potential coprognosticator to the acute patient physiologic and laboratory evaluation (APPLE) score in cats. ANIMALS: One hundred nineteen cats admitted to intensive care units (ICU) and 13 healthy cats at 2 university teaching hospitals. METHODS: Prospective study. Clinical examinations were performed, APPLE scores calculated, and serum cTnI and serum amyloid A (SAA) measured within 24 hours after admission. Outcome was defined as death/euthanasia or survival to discharge, 28 and 90 days after ICU-admission. Prognostic capacity of cTnI, APPLE scores and models combining cTnI and scores were evaluated by receiver-operator-characteristic analyses. RESULTS: Median (IQR) serum cTnI concentration was higher in ill (0.63 [0.18-2.65] ng/mL) compared to healthy (0.015 [0.005-0.041] ng/mL) cats (P < .001) and higher in subgroups with structural cardiac disease (2.05 [0.54-16.59] ng/mL; P < .001) or SAA >5 mg/L (0.84 [0.23-2.81] ng/mL; P = .009) than in cats without these characteristics (0.45 [0.12-1.70] and 0.35 [0.015-0.96] ng/mL). The in-hospital case fatality rate was 29%. Neither serum cTnI concentration for all critically ill cats (area-under-the-curve 0.567 [95% CI 0.454-0.680], n = 119) or subgroups (0.625 [0.387-0.863], n = 27; 0.506 [0.360-0.652], n = 86), nor APPLE scores (fast 0.568 [0.453-0.682], full 0.585 [0.470-0.699], n = 100), were significant prognosticators for death. CONCLUSIONS AND CLINICAL IMPORTANCE: Abnormally high serum cTnI concentration was common in critically ill cats. Unlike in dogs, cTnI did not confer prognostic information regarding death.

A Questionnaire Survey on Long-Term Outcomes in Cats Breed-Screened for Feline Cardiomyopathy.

Feline cardiomyopathy (FCM) is an important contributor to feline morbidity and mortality. This explorative follow-up questionnaire study was aimed at investigating the long-term outcome in cats breed-screened for FCM (BS-FCM) in three Nordic countries. Records of cats with ≥1 BS-FCM between 2004−2015 were included. Of the 1113 included cats, 104/1113 (9.3%) had developed FCM at some time-point. Fifty-nine of the 104 (56.7%) FCM cats were diagnosed within the screening program (ScreenFCM), and 33/59 (55.9%) of these were diagnosed at the first BS-FCM. ScreenFCM cats or with an owner-reported FCM diagnosis at a later time-point had a higher risk of cardiac-related death compared to cats that never developed FCM. A shorter lifespan was found in ScreenFCM cats compared to those with normal screen results (p < 0.001). Times to all-cause mortality were shorter (p < 0.001) in cats that developed FCM at any time-point compared to those that did not. Non-cardiac morbidities were similar in all screen classification groups. The large proportion of cats that developed FCM at a later time-point underscores the need for repeated screenings later in life. Cats that developed FCM at any time-point had a shorter lifespan, with a similar proportion and in line with the nature of non-cardiac morbidities, compared to those without FCM.

Repeatability of radiographic assessments for feline hip dysplasia suggest consensus scores in radiology are more uncertain than commonly assumed.

Variation in the diagnostic interpretation of radiographs is a well-recognised problem in human and veterinary medicine. One common solution is to create a 'consensus' score based on a majority or unanimous decision from multiple observers. While consensus approaches are generally assumed to improve diagnostic repeatability, the extent to which consensus scores are themselves repeatable has rarely been examined. Here we use repeated assessments by three radiologists of 196 hip radiographs from 98 cats within a health-screening programme to examine intra-observer, inter-observer, majority-consensus and unanimous-consensus repeatability scores for feline hip dysplasia. In line with other studies, intra-observer and inter-observer repeatability was moderate (63-71%), and related to the reference assessment and time taken to reach a decision. Consensus scores did show reduced variation between assessments compared to individuals, but consensus repeatability was far from perfect. Only 75% of majority consensus scores were in agreement between assessments, and based on Bayesian multinomial modelling we estimate that unanimous consensus scores can have repeatabilities as low as 83%. These results clearly show that consensus scores in radiology can have large uncertainties, and that future studies in both human and veterinary medicine need to include consensus-uncertainty estimates if we are to properly interpret radiological diagnoses and the extent to which consensus scores improve diagnostic accuracy.

The TNNT2:c.95-108G>A variant is common in Maine Coons and shows no association with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common and potentially fatal heart disease in many cat breeds. An intronic variant in TNNT2, c.95-108G>A, was recently reported as the cause of HCM in the Maine Coon. The aim of this study was to determine this variant's allele frequency in different populations and its possible association with HCM. Based on 160 Maine Coon samples collected in Belgium, Italy, Sweden and the USA, the variant's allele frequency was estimated to be 0.32. Analysis of the 99 Lives feline whole genome sequencing database showed that the TNNT2 variant also occurs in other breeds, as well as mixed-breed cats. Comparison of 31 affected and 58 healthy cats did not reveal significantly increased odds for HCM in homozygotes. Based on the combined evidence and in agreement with the standards and guidelines for the interpretation of sequence variants, this variant is currently classified as a variant of unknown significance and should not be used for breeding decisions regarding HCM.

Correction: The genetic consequences of dog breed formation-Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels.

[This corrects the article DOI: 10.1371/journal.pgen.1009726.].

The genetic consequences of dog breed formation-Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels.

Selective breeding for desirable traits in strictly controlled populations has generated an extraordinary diversity in canine morphology and behaviour, but has also led to loss of genetic variation and random entrapment of disease alleles. As a consequence, specific diseases are now prevalent in certain breeds, but whether the recent breeding practice led to an overall increase in genetic load remains unclear. Here we generate whole genome sequencing (WGS) data from 20 dogs per breed from eight breeds and document a ~10% rise in the number of derived alleles per genome at evolutionarily conserved sites in the heavily bottlenecked cavalier King Charles spaniel breed (cKCs) relative to in most breeds studied here. Our finding represents the first clear indication of a relative increase in levels of deleterious genetic variation in a specific breed, arguing that recent breeding practices probably were associated with an accumulation of genetic load in dogs. We then use the WGS data to identify candidate risk alleles for the most common cause for veterinary care in cKCs-the heart disease myxomatous mitral valve disease (MMVD). We verify a potential link to MMVD for candidate variants near the heart specific NEBL gene in a dachshund population and show that two of the NEBL candidate variants have regulatory potential in heart-derived cell lines and are associated with reduced NEBL isoform nebulette expression in papillary muscle (but not in mitral valve, nor in left ventricular wall). Alleles linked to reduced nebulette expression may hence predispose cKCs and other breeds to MMVD via loss of papillary muscle integrity.

A novel canine reference genome resolves genomic architecture and uncovers transcript complexity.

We present GSD_1.0, a high-quality domestic dog reference genome with chromosome length scaffolds and contiguity increased 55-fold over CanFam3.1. Annotation with generated and existing long and short read RNA-seq, miRNA-seq and ATAC-seq, revealed that 32.1% of lifted over CanFam3.1 gaps harboured previously hidden functional elements, including promoters, genes and miRNAs in GSD_1.0. A catalogue of canine "dark" regions was made to facilitate mapping rescue. Alignment in these regions is difficult, but we demonstrate that they harbour trait-associated variation. Key genomic regions were completed, including the Dog Leucocyte Antigen (DLA), T Cell Receptor (TCR) and 366 COSMIC cancer genes. 10x linked-read sequencing of 27 dogs (19 breeds) uncovered 22.1 million SNPs, indels and larger structural variants. Subsequent intersection with protein coding genes showed that 1.4% of these could directly influence gene products, and so provide a source of normal or aberrant phenotypic modifications.

Demography, heritability and genetic correlation of feline hip dysplasia and response to selection in a health screening programme.

Feline hip dysplasia (FHD) is a debilitating condition affecting the hip joints of millions of domestic cats worldwide. Despite this, little is known about FHD except that it is relatively common in the large breed Maine Coon. We used 20 years of data from 5038 pedigree-registered Maine Coon cats in a radiographic health screening programme for FHD to determine, for the first time, its heritability, genetic correlation to body mass and response to selection. FHD prevalence was 37.4%, with no sex predilection; however, FHD severity increased with age and body mass. Heritability of the radiographic categories used to classify FHD severity was 0.36 (95%CI: 0.30-0.43). The severity of FHD symptoms was also genetically correlated with body mass (0.285), suggesting that selection for a large body type in this breed concurrently selects for FHD. Support for this was found by following generational responses to selective breeding against FHD. Not only did selective breeding successfully reduce the severity of FHD symptoms in descendants, but these cats were also smaller than their ancestors (-33g per generation). This study highlights the value of breeding programmes against FHD and cautions against breed standards that actively encourage large bodied cats.

In vitro bioanalysis of drinking water from source to tap.

The presence of chemical pollutants in sources of drinking water is a key environmental problem threatening public health. Efficient removal of pollutants in drinking water treatment plants (DWTPs) is needed as well as methods for assessment of the total impact of all present chemicals on water quality. In the present study we have analyzed the bioactivity of water samples from source to tap, including effects of various water treatments in a DWTP, using a battery of cell-based bioassays, covering health-relevant endpoints. Reporter gene assays were used to analyze receptor activity of the aryl hydrocarbon receptor (AhR), estrogen receptor (ER), androgen receptor (AR), peroxisome proliferator-activated receptor alpha (PPARα) and induction of oxidative stress by the nuclear factor erythroid 2-related factor 2 (Nrf2). DNA damage was determined by Comet assay. Grab water samples were concentrated by HLB or ENV solid phase extraction and the water samples assayed at a relative enrichment factor of 50. The enrichment procedure did not induce any bioactivity. No bioactivity was detected in Milli-Q water or drinking water control samples. Induction of AhR, ER and Nrf2 activities was revealed in source to tap water samples. No cytotoxicity, PPARα or AR antagonist activity, or DNA damage were observed in any of the water samples. A low AR agonist activity was detected in a few samples of surface water, but not in the samples from the DWTP. The treatment steps at the DWTP, coagulation, granulated activated carbon filtration, UV disinfection and NH2Cl dosing had little or no effect on the AhR, Nrf2 and ER bioactivity. However, nanofiltration and passage through the distribution network drastically decreased AhR activity, while the effect on Nrf2 activity was more modest and no apparent effect was observed on ER activity. The present results suggest that bioassays are useful tools for evaluation of the efficiency of different treatment steps in DWTPs in reducing toxic activities. Bioassays of AhR and Nrf2 are useful for screening of effects of a broad range of chemicals in drinking water and ER activity can be monitored with a high sensitivity.

Mixture effects of dietary flavonoids on steroid hormone synthesis in the human adrenocortical H295R cell line.

Humans are exposed to a mixture of dietary flavonoids with a variety of potential beneficial and harmful effects. Flavonoids are endocrine disruptors, acting both at receptor level and by interfering with steroid hormone synthesis. Due to a high dietary intake and the potential to cause mixture effects, assessment of combined exposure of flavonoids is required. We have studied effects on cortisol, aldosterone, testosterone and oestradiol secretion of the individual isoflavones daidzein and genistein, the flavone apigenin and the mixture of the three flavonoids in human adrenocortical H295R cells. The most vulnerable targets of the flavonoids were the secretion of cortisol and testosterone, which were inhibited by daidzein and genistein with IC50 values below 1 µM. An equimolar mixture of the flavonoids caused inhibition of cortisol, aldosterone and testosterone secretion in an additive manner. The observed mixture effect was described well by both concentration addition (CA) and independent action (IA) prediction models. Both prediction models underestimated the effect on oestradiol secretion. We conclude that the three flavonoids exhibit specific effects on steroid hormone secretion. A mixture of the flavonoids caused additive effects emphasizing the need to assess flavonoids together as a group. The prediction models are valuable tools for mixture assessment.

Mixture effects of imidazole fungicides on cortisol and aldosterone secretion in human adrenocortical H295R cells.

Exposure to chemicals commonly occurs in the form of mixtures. Methods and models are required to analyze and predict the effect of mixtures in order to improve risk assessment. The steroidogenesis and hormone production of the adrenal gland is a sensitive target for endocrine-disrupting chemicals including imidazoles. Here, we exposed human adrenocortical H295R cells to the individual imidazole fungicides prochloraz, ketoconazole, imazalil and their mixtures and analyzed the effects on secretion of cortisol and aldosterone and the effects on steroidogenic gene expression. The individual imidazole fungicides prochloraz, ketoconazole and imazalil and their mixtures inhibited cortisol secretion in a similar monotonic dose-response pattern with an EC(50) value of approximately 0.1 microM. Aldosterone secretion, in contrast, displayed a biphasic dose-response, with low-dose stimulation of up to maximum twofold and high-dose inhibition. Biphasic dose-responses were found following prochloraz and ketoconazole exposure and their mixtures, but not following imazalil exposure. The inhibition of cortisol secretion was equally well predicted with the concentration addition (CA) and independent action (IA) models, while the biphasic aldosterone response was partially predicted by a modified CA model and not predicted well by a modified IA model. Changes in expression levels of steroidogenic genes could not conclusively explain the different effects on the two hormone endpoints or the different specificities of the imidazoles. We conclude that single imidazoles and mixtures have specific effects on adrenal hormone secretion. These effects can only partly be predicted using current models and need to be further analyzed in terms of in vivo relevance and human risk assessment.

A biphasic effect of the fungicide prochloraz on aldosterone, but not cortisol, secretion in human adrenal H295R cells--underlying mechanisms.

The widely used imidazole fungicide prochloraz displays anti-androgenic effects partly via inhibition of testicular steroidogenesis and testosterone secretion. Adrenal steroidogenesis and hormone secretion may also be a target of this endocrine disruptor. Herein, we demonstrate a dose-dependent inhibition of cortisol secretion and a biphasic effect on aldosterone secretion, with a 2-fold stimulation at low concentrations and a strong inhibition at high concentrations, following prochloraz treatment (0-10 microM) of human adrenocortical H295R cells. Analysis of the dose-dependent effects of prochloraz on the secretion of steroidogenic intermediates suggested that the observed effects on cortisol and aldosterone secretion might be mediated by inhibition of the steroidogenic steps catalysed by CYP17A1 and CYP21A2. The inhibition of CYP17A1 was reflected on the level of expression of steroidogenic genes as analysed by quantitative RT-PCR. In addition, analysis of enzyme activity showed a dose-dependent inhibitory effect of prochloraz on the activity of CYP17A1 and CYP21A2, but not CYP11B1. We have demonstrated specific effects of prochloraz on adrenal steroidogenic pathways and hormone secretion via inhibition of steroidogenic CYP enzymes. The disruption of adrenal hormone secretion may result in altered endocrine homeostasis and affect human health.

Secretion of cortisol and aldosterone as a vulnerable target for adrenal endocrine disruption - screening of 30 selected chemicals in the human H295R cell model.

The adrenal gland is a vulnerable target for toxic insult. Disruption of adrenal steroidogenesis and hormone secretion may cause serious effects on human health. A human in vitro model is needed to predict effects, and elucidate mechanisms of endocrine disruption and adrenal toxicity. The human adrenocortical cell line H295R has been used to screen for effects on sex hormones. Here, we have analyzed the effect of 30 potential endocrine disrupting chemicals on the secretion of cortisol and aldosterone from the H295R cells, using specific ELISA assays. The effect of chemicals was analyzed for basal and forskolin- or angiotensin II-stimulated hormone secretion. The chemicals were tested at the highest concentration where they displayed no evident unspecific cytotoxicity. Quantitative and qualitative differences in effects on hormone secretion were demonstrated for the various chemicals. A subset of the chemicals displayed different effects on cortisol and aldosterone secretion, and in some cases the effects were different between basal and stimulated hormone secretion. Aminoglutethimide, prochloraz, ketoconazole, 6-hydroxyflavone, imazalil and etomidate had the most marked inhibitory effects on cortisol (with or without forskolin) and ketoconazole, 6-hydroxyflavone, imazalil and etomidate had the most marked effects on aldosterone (with or without angiotensin II). The results are discussed in terms of known effects, structural similarity and possible mechanisms. We have shown that adrenal steroidogenesis is a vulnerable target for toxic insult and that the H295R assay is a useful in vitro model for screening purposes.